1.1 Phytochemicals study of K. parviflora

Flavones, which consist of multiple methoxylated substituents, specifically PMFs, are the major components in K. parviflora, followed by acetophenone, chalcone derivatives, kaempferiaosides, and other phenolic compounds. To date, 15 major PMFs have been successfully identified, and their respective biological activities are summarized in Table 1. The methoxy group in the chemical structure varied from position C-3, C-3′, C-4′, C-5, and C-7; meanwhile for hydroxy group (C-5, C-3′ & C4′).

Table 1 PMFs in K. parviflora and their respective biological activities.

Chemical structure	Name	Biological activities
	5,7-Dimethoxyflavone	Anti-inflammatory (Lee et al., 2022) Anticancer (Kim et al., 2018) Alzheimer’s (Youn et al., 2016) Antioxidant (Thao et al., 2016) Antidiabetic (Kobayashi et al., 2015) Anti-acetylcholinesterase (Sawasdee et al., 2009)
	5,7,4′-Trimethoxyflavone	Antibacterial (Sookkhee et al., 2022) Anti-inflammatory (Phung et al., 2021) Alzheimer’s (Natsume et al., 2020) Antioxidant (Thao et al., 2016) Anti-acetylcholinesterase (Seo et al., 2017) Antiglycation (Nakata et al., 2014) Anticancer (Hossain et al., 2012) Antidiabetic (Azuma et al. 2011) Antimycobacterial (Yenjai et al., 2004) Antifungal (Yenjai et al., 2004) Anti-parasites (Yenjai et al., 2004)
	3,5,7-Trimethoxyflavone	Antibacterial (Sookkhee et al., 2022) Antioxidant (Thao et al., 2016) Anti-inflammatory (Sae-Wong et al., 2011) Antifungal (kummee et al., 2008) Antiallergy (Tewtrakul et al., 2008)
	5,7,3′,4′-Tetramethoxyflavone	Anti-inflammatory (Ongchai et al., 2021) Alzheimer’s (Natsume et al., 2020) Antidiabetic (Azuma et al., 2011) Anti-parasites (Yenjai et al., 2004)
	3,5,7,4′-Tetramethoxyflavone	Antioxidant (Thao et al., 2016) Anti-inflammatory (Toda et al., 2016a) Antiglycation (Nakata et al., 2014) Antidiabetic (Horikawa et al., 2012) Anticancer (Hossain et al., 2012) Antiallergy (Tewtrakul et al., 2008) Antimycobacterial (Yenjai et al., 2004) Antifungal (Yenjai et al., 2004)
	3,5,7,3′,4′-Pentamethoxyflavone	Antibacterial (Sookkhee et al., 2022) Anticancer (Kim et al., 2018) Anti-acetylcholinesterase (Seo et al., 2017) Alzheimer’s (Youn et al., 2016) Anti-inflammatory (Jakhar et al., 2014) Antiglycation (Nakata et al., 2014) Antidiabetic (Okabe et al., 2014) Antioxidant (Jakhar et al., 2014)
	5-Hydroxy-7-Methoxyflavone	Anticancer (Sun et al., 2021) Antioxidant (Thao et al., 2016) Antidiabetic (Shimada et al., 2011) Anti-inflammatory (Tewtrakul et al., 2009) Antiallergy (Tewtrakul et al., 2008)
	5-Hydroxy-7,4′-Dimethoxyflavone	Antioxidant (Thao et al., 2016) Anti-inflammatory (Horigome et al., 2014) Antidiabetic (Shimada et al., 2011) Antiallergy (Tewtrakul et al., 2008)
	5-Hydroxy-3,7-Dimethoxyflavone	Antioxidant (Thao et al., 2016) Anti-inflammatory (Toda et al., 2016a) Antidiabetic (Shimada et al., 2011) Antiallergy (Tewtrakul et al., 2008)
	5-Hydroxy-7,3′,4′-Trimethoxyflavone	Anti-allergy (Kobayashi et al., 2015)
	5-Hydroxy-3,7,4′-Trimethoxyflavone	Antioxidant (Thao et al., 2016) Anti-inflammatory (Toda et al., 2016a) Antiallergy (Kobayashi et al., 2015) Antidiabetic (Shimada et al., 2011)
	5-Hydroxy-3,7,3′,4′-Tetramethoxyflavone	Anti-inflammatory (Lee et al., 2022) Antioxidant (Thao et al., 2016) Anticancer (Hossain et al., 2012) Antiallergy (Tewtrakul et al., 2008)
	5,3′-Dihydroxy-3,7,4′-Trimethoxyflavone	Antiallergy (Kobayashi et al., 2015) Anti-inflammatory (Sae-Wong et al., 2011)
	5,4′-Dihydroxy-7-Methoxyflavone	Antioxidant (Thao et al., 2016) Antidiabetic (Kobayashi et al., 2015)
	4′-Hydroxy-5,7-Dimethoxyflavone	Antioxidant (Thao et al., 2016) Antiallergy (Kobayashi et al., 2015)

3.1 In-vivo toxicity study

Although KPE has been used in traditional remedies for centuries, no toxicity guidelines have been utilized throughout its consumption. The safety profiles of K. parviflora in various doses were discussed in toxicology in vivo studies on rats from 2006 to 2019. While no optimal amounts have been confirmed, intake of 2.0 g/kg body weight of K. parviflora was categorized as safe based on a 7-day acute single-oral toxicity study, with low toxicity levels recorded (Sae-wong et al., 2009). The results were within the satisfactory range given that in another 14-day toxicity test, the approximate lethal dose (ALD) recorded was more than 5.0 g/kg, with no apparent difference in body weight changes for both female and male Sprague-Dawley rats (SD rats) (Han & Park, 2018). However, contradicting the earlier report, a 6-month chronic toxicity test of high intake of KPE at 500 mg/kg body weight /day revealed a significant decline in male rats’ body weight starting from week 8 of treatment, possibly due to lower food consumption. Meanwhile, for female rats, a notable surge was visible in glucose and cholesterol levels at a higher dose (174.96 & 116.18 mg/dL) compared to the control, 141.59 and 68.93 mg/dL, respectively, which may be attributed to overdose with a high level of ALT and BUN level detected, a sign of kidney and liver disease (Chivapat et al., 2010). Besides, the platelet count of female rats in low doses of K. parviflora (5 and 50 mg/kg /day) was decreased (880.5 × 10³/mm³) compared to the control study (932.0 × 10³/mm³). In contrast, increases in platelet counts were recorded with consumption of 25 mg/kg /day doses in 90-day hematological analysis (100.4 × 10¹²/L) against the control groups (82.8 × 10¹²/L); however, no apparent difference in body weight changes, glucose, and cholesterol level for female and male rats recorded in this study (Yoshino et al., 2019). Meanwhile, in other hematological parameters analysis, K. parviflora remained non-toxic to both female and male rats in various doses, and no adverse effect was reported on red blood cell count, white blood cell count, Hemoglobin count, and hematocrit count, in agreement with the initial findings (Sudwan et al., 2006). Meanwhile, consumption of 100 mg/kg body weight of K. parviflora twice a day does not exert any changes on the level of kidney and liver enzymes; creatinine, blood urea nitrogen, alkaline phosphatase (ALP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT), which supported by other toxicology studies (Yorsin et al., 2014). However, at high doses (249 mg/kg/day), the creatinine level was lowered to 24.75 mmol/L, while ALP observed a higher count (291.8 U/L) than the control. Nevertheless, the changes were noticeable yet within the optimum range in regulating kidney and liver functions (Yoshino et al., 2019).

3.2 In vitro toxicity study

On another note, the toxicity effect of crude extract and its PMFs were investigated on normal cell lines to determine its selectivity activity and optimum concentration level. Aqueous extract of crude K. parviflora showed low toxicity on normal Madin-Darby canine kidney (MDCK) cells with an IC₅₀ value of 468.2 μg/ml. However, ethanolic extract's toxicity was unexpectedly higher, with an IC₅₀ value of 2.2 μg/ml (Sornpet et al., 2017). In another study, at the highest concentration of KPE (200 μg/ml), the normal human epidermal keratinocytes (HaCaT) cell growth was maintained at 90 % (Wang et al., 2022). In another study with similar types of cells, no toxicity was observed in the cell when administered with crude extract at a concentration of 400 μg/ml (Lee et al., 2018). All 6 PMFs compounds isolated from the crude extract exhibit no toxicity up to 100 µM concentration (Lee et al., 2022). Meanwhile, the crude extract demonstrated high selectivity against normal human umbilical vein endothelial cells (HUVEC) cells, with low toxicity recorded after 24 h (IC₅₀: 88.85 μg/ml) (Tangjitjaroenkun et al., 2021). Treatment of up to 100 μg/ml of crude extract does not exhibit significant toxicity against normal human dermal fibroblast cells (HDF), with cell growth maintained above 90 % (Sitthichai et al., 2022). In another toxicity study of the isolated compound from KPE, the HDF cell viability retained above 90 % when treated up to 100 µM of compound 3,5,7,3′,4′-Pentamethoxyflavone (PeMF), followed by 5,7-DMF (25 µM) and 5,7,4′-TMF (25 µM). In another study, the reduction of normal fibroblast HS68 cells from cellular senescence was suppressed with increased cell growth by the crude extract up to 10 µM concentration (Park et al., 2017). The in vitro study of crude KPE and its isolated compounds demonstrate potent inhibition of normal cell death.

4.1 KPE inhibits inflammatory markers

Inhibition of cytokines is a primary pathway to suppress inflammatory release. In the study by Nemidkanam et al. (2020), the mRNA expression of IL-8 was significantly suppressed at 16 μg/ml crude extract by 2.3-fold and 2.07-fold after 6 and 12 h, respectively. Remarkably, the secretion was also reduced to 57.68 pg/ml and 72.15 pg/ml in the same period, respectively, compared to control. Besides, crude extract with a concentration range of 10 ng/ml significantly inhibits the expression and release of IL-6, IL-1β, and TNF-α in a dose-controlled manner (Table 2) (Ongchai et al., 2021). The crude extract at 1000 μg/ml effectively decreased the expression of these cytokines by 7 %, 39 %, and 57 %, respectively (Horigome et al., 2017). Mitogen-activated protein kinase (MAPK) family comprises proteins ERK, p38, JNK, and protein kinase B (PKB/Akt), which are predominantly associated with the activation of many inflammatory mediators. The crude extract potently suppressed MAPK proteins (Table 2) (Lee et al., 2018), with phosphorylation of ERK, p38, and JNK reduced by 29.8 %, 40.6 %, and 39.0 %, respectively (Ongchai et al., 2021). Furthermore, 15 μg/ml of KPE also downregulated the activity of Nuclear factor-κB (NF-κB) and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) by 3 and 0.97-fold, respectively (Takuathung et al., 2021). Meanwhile, 3 h of treatment significantly suppressed activator protein 1 (AP-1) activity, which is responsible for elevated COX-2 expression (Lee et al., 2018).

As mentioned in Section 2., MMPs are an inflammatory modulator regulating various inflammatory markers. The activity of MMPs is essential in various cell biological and physiological processes, including tissue remodeling, such as wound healing, angiogenesis, and bone development. Nonetheless, it’s associated with multiple inflammatory-mediated diseases such as arthritis, sclerosis, diabetes, and cancer progression (Chen et al., 2017). In a study by Kobayashi et al. (2018), low concentration of KPE (20 µg/ml) potently suppressed expression of IL-1β-induced inflammation enzyme MMP-1 (0.74-fold), MMP-3 (0.82-fold) and MMP-13 (0.95-fold) activity, compared to control (1.00-fold). In another study in vivo, 100 and 200 mg/kg/day of K. parviflora potently suppressed the expression of MMPs (MMP-2, MMP-3, MMP-9 & MMP-13) in a concentration-controlled behaviour, compared to the control (Park et al., 2014).

ROS regulates inflammatory markers in various stages of inflammatory responses (Chelombitko, 2018). Numerous studies discovered flavonoids with potent antioxidants capable of scavenging ROS free radicals from oxidative damage and reducing oxidative stress (Liu et al., 2018). KPEs were reported to reduce the ROS level by 13 % between 1 and 10 μg/ml and 67.6 % at 1000 μg/ml (Park et al., 2017; Horigome et al., 2017). Additionally, KPE at 20 μg/ml (Jin and Lee, 2018) and up to 1000 μg/ml show potent suppression of COX-2 expression. However, at low concentrations (5 & 10 μg/ml), COX-2 suppression was ineffective (Horigome et al., 2014). Astonishingly, 15 μg/ml of the crude extract can reduce COX-2 expression and secretion by 18-fold and 30 %, respectively (Table 2) (Takuathung et al., 2021). In an in vivo study, consuming 50 and 100 mg/kg body weight significantly suppressed COX-2 expression (Lee et al., 2018). Meanwhile, the crude extract inhibited PGE2 released by COX-2 in JB6 P + cells in a concentration-dependent manner (Lee et al., 2018). The extract suppressed the PGE2 level in macrophage cells with an IC₅₀ value of 9.2 µg/ml (Sae-wong et al., 2009). Additionally, treatment with KPE reduced the expression of iNOS and NO in a concentration-controlled manner (Jin and Lee, 2018; Kongdang et al., 2019). The K. parviflora extract at concentrations of 3.75 and 15 μg/mL increased the iNOS expression by 6- and 18-fold, while NO levels decreased to 45 and 30 μM, respectively (Takuathung et al., 2021)., in another study, iNOS expression was unchanged. Nonetheless, NO production was significantly suppressed (IC₅₀: 482.6 μg/ml) (Horigome et al., 2017).

4.2 Isolated compound of KPE inhibits MAPK and AKT proteins

Treatment of 50 & 100 µM 3,5,7-Trimethoxyflavone (3,5,7-TMF) significantly suppressed MAPK proteins with reduction of ERK (1.30 & 0.77-fold), JNK (4.88 & 4.86-fold), p38 (5.58 & 5.20-fold) and AKT protein phosphorylation by 4.88 & 4.86-fold, respectively (Table 2) (Lee et al., 2022). Similarly, 5,7,4′-TMF possesses a comparable effect at lower concentrations (6.25 & 12.5 µM) (Phung et al., 2021). However, the inconsistent result was observed in RAW 264.7 cells with 5,7,4′-TMF, and 5,7-DMF only demonstrated moderate suppression on phosphorylation of JNK and ERK (Sae-Wong et al., 2011); nonetheless, 5,7-DMF was capable of alleviating AKT phosphorylation (Kim & Hwang, 2020).

4.3 Isolated compound of KPE inhibits MMPs activities

At 50 µM, 3,5,7-TMF attenuated MMP-1 mRNA expression and secretion in HDF cells by 2.53- and 1.99-fold, respectively (Lee et al., 2022). Compound 5,7-DMF, 5,7,4′-TMF, and PeMF show potent activity in suppressing the MMP-1 gene, ranging from 34 % to 47 %. Long-term treatment of these PMFs contributes to the reduction of MMP-1 activity by 68.57 %, 71.43 %, and 28.57 %, respectively (Table 2) (Klinngam et al., 2022).

4.4 Isolated compound of KPE as oxidative stress suppressor

Other than that, ROS, COX-2, and HO-1 expression were significantly suppressed by 3,5,7-TMF in a dose-dependent behaviour, as shown in Table 2. At 50 and 100 µM, ROS expression was suppressed by 1.88-fold and 1.33-fold, COX-2 (4.78 and 2.36-fold), and HO-1 (1.68 and 3.03-fold) (Lee et al., 2022). Likewise, 5,7,4′-TMF concomitantly suppressed COX-2 and ROS expression at lower concentrations (Phung et al., 2021), with ROS level reduced by 40 % at the lowest concentration (3 μg/ml) (Klinngam et al., 2022). In addition, a strong inhibitory effect was demonstrated by 5-hydroxy-3,7,3′,4′-Tetramethoxyflavone (5H-3,7,3′,4′-TeMF) with 102 % suppression on COX-2 level (Horigome et al., 2017) and significant inhibition on PGE2 level with IC₅₀ of 16.3 µM (Tewtrakul et al., 2009). Inhibition of protein transcription factors like NF-κB and AP-1 complexes capable of reducing oxidative stress. Isolated K. parviflora, 5,7,4′-TMF significantly reduced the expression of these complexes in a dose-controlled manner (Park et al., 2014). Meanwhile, 5,7-DMF also exerts similar suppression activity towards the phosphorylation of IκB-α and NF-κB (Table 2) (Jin and Lee, 2018).

4.5 Isolated compound of KPE suppressed inflammatory cytokines mediators

Pro-inflammatory cytokines are involved in elevating inflammation processes. Compound 5,7,4′-TMF, 3,5,7-TMF, 5,7-DMF, and PeMF show potent suppression of IL-1β and IL-6 mRNA expression and released in a dose-controlled manner (Phung et al., 2021), with 5,7-DMF, demonstrated the most potent effect (Ongchai et al., 2021). Compound 5,7,4′-TMF alleviate secretion of IL-1β and IL-6 at low concentration (12.5 µM) by 1.03 and 1.12-folds, respectively (Lee et al., 2022). Meanwhile, in another study, 5,7-DMF (50 µM) only reduced it by 2 % and 4 %, respectively (Horigome et al., 2017), while no change was observed in IL-1β when treated with PeMF (Kongdang et al., 2019). In comparison, 50 µM of 5H-TeMF significantly suppressed the expression of IL-6, IL-1β, and TNF-α with reductions of 73 %, 64 %, and 36 %, respectively (Horigome et al., 2017). Compound 5,7-DMF and 5H-3,7,3′,4′-TeMF demonstrate potent suppression of TNF- α expression with 100 % inhibition in antigen-induced RBL-2H3 cells (Horigome et al., 2014). In human THP-1 monocytes, 5,7-DMF reduced TNF-α released by 56.89 % (Klinngam et al., 2022). Meanwhile, in RAW 264.7 cells, 5,7,4′-TMF, 5,7-DMF, and 5,7,3′,4′-TeMF moderately suppressed TNF- α secretion with an IC₅₀ value of 30 to 100 µg/ml (Sae-Wong et al., 2011). In contrast, consumption of 25 and 50 mg/kg/day of 5,7-DMF potently suppressed the release of IL-6 and TNF-α levels and downregulated NF-κB expression (Kim & Hwang, 2020).

4.6 Isolated compound of KPE inhibits iNOS and NO activities

Nitric oxide (NO) involves inflammation's pathogenesis with three major isotypes: iNOS, eNOS, and iNOS (Sierra et al., 2014). iNOS is notably expressed in macrophages and microglia during cell damage induced by inflammatory markers (Brown & Neher, 2010). Meanwhile, overexpression of NO in cytokines-mediated macrophages is a primary factor contributing to inflammatory progression with the presence of NOSs (Sharma et al., 2007). In a study of Propionibacterium acnes-induced iNOs, it was reported that 5,7-DMF and 5H-3,7,3′,4′-TeMF reduced the expression of iNOS level significantly in a concentration-controlled manner (Jin and Lee, 2018; Sae-wong et al., 2009). In contrast, lipopolysaccharides (LPS)-induced iNOS mRNA expression was weakly suppressed by 5,7-DMF and 5H-TeMF in RAW264.7 cells (Horigome et al., 2017). Although weak LPS-induced iNOS expression was reported by 5H-TeMF, the compound shows the highest NO inhibitory effect (IC₅₀ = 16.1 µM) (Tewtrakul and Subhadhirasakul, 2008; Tewtrakul et al., 2009). Besides, 5,7-DMF remarkably suppressed LPS-induced NO level with the lowest IC₅₀ of 9.03 µM (Fuchino et al., 2018), followed with 5,7,4′-TMF and 5,7,3′,4′-TeMF, both recorded IC₅₀ values of 14.2 µM and 16.6 µM, respectively (Sae-Wong et al., 2011). A similar effect was observed in human umbilical vein endothelial cells (HUVEC) (Horigome et al., 2017). Nevertheless, 3,5,7-TMF shows weak NO inhibitory activity with IC₅₀ of 44 to 60 µg/ml (Sae-Wong et al., 2011). Meanwhile, PeMF at 25, 50, and 100 µg/ml concentration inhibit NO activity by 17.57 %, 21.72 %, and 31.67 %, respectively. However, the result is lower than quercetin, which shows 53.2 % inhibition at 100 µg/ml concentration (Jakhar et al., 2014).

5.1 Anticancer activity of PMFs isolated from KPE

Isolated PMFs from KPE potently induced apoptotic cell death in a dose-dependent behaviour. Kim et al. (2018) reported that compound 5,7,4′-TMF (50 µM) demonstrated the most potent inhibitory effect on gastric cancer cells, SNU-16, with almost 50 % cell growth reduction. In the same study, other PMFs, like 5,7-DMF and PeMF, required at least 100 µM to obtain similar results (Kim et al., 2018). Compound 5,7,4′-TMF induced SNU-16 cell death in the apoptotic pathway, with the percentage of cell accumulation in the sub-G-1 phase increased in a dose-controlled manner, from 3.9 % to 35.1 % at 50 µM. Meanwhile, 5-hydroxy-7-methoxyflavone (5H-7-MF) induced apoptosis in pancreatic cancer cells, PANC-1, through cleavage in caspase-3 (Sun et al., 2021). On another note, 5,7,4′-TMF, 5H-3,7,3′,4′-TeMF, and 3,5,7,4′-TeMF induced cytotoxicity on human colorectal carcinoma cells (HCT15) in the concentration range from 25 to 500 µM. Among these PMFs, 5,7,4′-TMF significantly reduced cell growth by almost 80 % at an optimal concentration of 100 µM. Meanwhile, the HCT15 cell growth was weakly suppressed by 5H-3,7,3′,4′-TeMF and 3,5,7,4′-TeMF with 29.2 % and 16.1 % inhibition, respectively, which may be due to the position of methoxy groups (Hossain et al., 2012). The result suggests that 5,7,4′-TMF shows the most potent anticancer activity by inducing apoptotic cell death. The compound stimulates cell death via four major apoptotic pathways, as confirmed via reverse-transcription polymerase chain reaction, RT-PCR (Kim et al., 2018). Firstly, the apoptotic effect of the PMFs was activated via suppression of the AKT/Mammalian target of rapamycin (mTOR)/Glycogen synthase kinase-3 beta (GSK3β) pathway. Suppressing AKT's phosphorylation reduced cancer cells' survival and mediated apoptotic protein activity. Secondly, a reduction in AKT-Phosphoinositide 3-kinases (PI3K) activity elevates the endoplasmic reticulum (ER) stress. These combinations overstimulate the C/EBP homologous protein (CHOP) gene in the ER-stress-stimulated apoptotic mechanism, resulting in higher activity on caspase-8 and −4, thus inducing apoptotic cell death (Siu et al., 2002). In addition, compound 5,7,4′-TMF was also reported to induce apoptotic cell death via extrinsic and intrinsic pathways. The extrinsic mechanism was stimulated by caspase-8 activation and cleavage on pro-apoptotic BH3 interacting-domain death agonist (Bid), thus activating the intrinsic pathway via oligomerization of Bcl-2-associated X protein (Bax), another pro-apoptotic protein. The PMFs stimulate increased Bax/BCl-2 and Bax/Bcl-xL ratio in a concentration-controlled behaviour, activating caspase-9 and −3 that initiate apoptosis cell death (Kim et al., 2018).

5.2 Anticancer activity of KPE

KPE within 1.00 mg/ml demonstrates potent cytotoxicity against ovarian cancer SKOV3 cells with a reduction of more than 70 % (IC₅₀: 0.53 mg/ml) (Table 3) (Paramee et al., 2018). The cancer cell doubling time improved to 32.6 h compared to untreated cancer cells (24 h) with 0.025 mg/ml extract before decreasing to 31.5 h at a higher concentration. Treatment with 0.05 mg/ml crude extract reduced the cell migration and invasion by almost 50 % and 70 %, respectively. In cancer treatment, MMP-2 and MMP-9 activity inhibition could minimize tumor invasion and metastasis. KPE reduced the activity of the MMPs in a concentration-controlled manner. At 0.01 mg/ml, the activity of MMP-9 and MMP-2 were mildly suppressed by 11.34 % and 7.48 % and improved to 31.17 % and 18.08 %, respectively, at higher concentrations. A similar result was observed in Hela 229 cells with suppression of MMP-2 activity by 30 % (IC₅₀: 0.22 mg/ml) (Potikanond et al., 2017). ERK and AKT activities are significantly involved in stimulating the cell death pathway. KPE at 0.01 mg/ml potently inhibits the phosphorylation of these proteins by 0.85 and 0.87-fold and 0.64 and 0.58-fold at 0.05 mg/ml, respectively. In similar concentrations, KPEs also demonstrated potent suppression of these proteins on Hela 229 cells (Potikanond et al., 2017) and HL-60 cells (Banjerdpongchai et al., 2008). The study shows that KPEs induced an apoptotic effect, with approximately 15.67 % and 26.33 % cell death at concentrations of 0.1 and 0.25 mg/ml, respectively. At higher concentrations (0.30 & 0.50 mg/ml), the percentage of a cell undergoing apoptosis significantly increased to 22.13 % and 41.13 %, respectively.

In cervical cancer, the crude extract significantly inhibits the proliferation of Hela 229 cells within the concentration range of 0.01 to 1 mg/ml. Maximum cytotoxicity effect was observed at 0.5 mg/ml with 90 % cell reduction (IC₅₀: 0.22 mg/ml) (Table 3). Cell treated with 0.3 and 0.5 mg/ml crude extract induced a higher percentage of apoptosis cell death with 39.8 % and 69.85 %, respectively, through cleavage on caspase-7 and caspase-9. In addition, 0.01, 0.05, and 0.1 mg/ml of KPE suppressed cell migration by 52.21 %, 63.23 %, and 84.54 %, respectively, and cell invasion (52.21, 42.17 and 78.12 %) (Potikanond et al., 2017). In the human urinary bladder cancer cell line (T24), treatment of ethanolic KPE for 1, 4, and 7 days induced potent cytotoxicity (IC₅₀: 29.62, 16.91, and 7.56 μg/ml) (Table 3). Meanwhile, in human prostate cancer cell lines (DU145, LNCaP & PC3), the crude extract significantly suppressed cell growth in a concentration-dependent manner. The mechanism of apoptotic cell death in prostate cancer was induced through a potent expression of the tumor protein p53 gene up to 4-fold via the intrinsic-mitochondria pathway (Tangjitjaroenkun et al., 2021).

In another study, KPE induced bile duct apoptotic cancer cell death (HuCCA-1 & RMCCA-1) with an IC₅₀ value of 46.13 and 61.97 µg/ml, respectively (Table 3) (Leardkamolkarn et al., 2009). A study by Banjerdpongchai et al. (2009 & 2008) demonstrates the potent anticancer activity of KPE on leukemic and HL-60 cancer cells, respectively. Treatment of KPE (24, 48, & 72 h) stimulates potent cytotoxicity on HL-60 cancer cells at IC₅₀ values of 25.5, 18.5, and 14.5 mg/ml, respectively (Table 3). Meanwhile, significant leukemic cancer cell (U937) reduction was also observed (IC₅₀: 92, 70, and 61 µg/ml). Both HL-60 and U937 cancer cells induced apoptotic cell death through cleavage on caspase-3 in similar crude extract concentration ranges. However, the crude extract triggered HL-60 cell death in the apoptotic pathway up to 80 mg/ml, followed by necrosis when treated at 100 mg/ml. Meanwhile, the crude extract demonstrates antiapoptotic roles on U937 cancer cells at low concentrations (10 and 20 µg/ml) based on the low mitochondrial transmembrane potential (MTP) recorded. Nonetheless, the antiapoptotic role of KPE was not thoroughly investigated as a study by Erster et al. (2004) and Padanilam (2003) demonstrated that the reduction of MTP may be attributed to changes in mitochondria membrane permeability by the release of other pro-apoptotic genes such as cytochrome c during the apoptotic process that modulated by Bcl-2 proteins. Furthermore, drug combinations between the crude extract and commercialized drug, paclitaxel, camptothecin, and doxorubicin significantly ameliorate the cell proliferation and cytotoxicity in a dose-controlled manner through an apoptotic effect on both leukemic and HL-60 cancer cells.

5.3 Anticancer activities of KPE via suppression of inflammatory mediators

Cancer cell proliferation and the progression of tumors occur through complex mechanisms and pathways, which include inflammatory mediators. Interestingly, expression of cytokines TNF-α and IL-6 activate signaling cascades and upregulation of protein kinases (MAPK, Akt, PI3k, ERK) and protein transcription (NF-kB & STAT3) eventually contribute to elevated levels of antiapoptotic protein (MCL-1), cancer cell proliferation, migration, and invasion in various organ system (Wang & Lin, 2008). Therefore, this review highlights the effective inhibition of pro-inflammatory mediators via modulating various signaling by KPE and isolated PMFs compound.

In this review, the anticancer activity of KPE was explored through the suppression of inflammatory mediators in Hela cervical cancer cells (Suradej et al., 2019) and ovarian carcinoma cancer cells (TOV-21G) (Thaklaewphan et al., 2021). In Hela cells, 7.5 and 15 µg/ml of KPE significantly inhibited the expression of IL-6 by 326.8 and 242.6-fold, respectively. A similar result was observed in TOV-21G, with the secretion reduced to 9077.78 and 6151.85 pg/ml when treated with 5 and 10 µg/ml crude extract, respectively. In addition, KPE also suppressed the phosphorylation of the transcription factors (NF-κB & STAT3). Nonetheless, no significant change was recorded on the NF-κB level in Hela cells. Treatment of 7.5 and 10 µg/ml of the crude extract significantly suppressed phosphorylation of protein kinase AKT in both cells, respectively. In TOV-21G cells, 10 µg/ml of KPE significantly suppressed phosphorylation of ERK and inhibited MCP-1 protein to 2.41-fold. In both cancer cells, deactivation of the inflammatory mediators significantly suppressed phosphorylation of antiapoptotic protein MCL-1 in a concentration-controlled manner, which induced apoptotic cell death. As a result, TOV-21G and Hela cell proliferation were potently reduced in a dose-controlled manner, with a 30 µg/ml IC₅₀ value recorded in TOV-21G; meanwhile, a significant reduction in cell growth was observed in Hela cells.

Previous studies reported that KPEs and PMFs potently reduced cancer cell proliferation and induced apoptosis cancer cell death through various pathways, including extrinsic pathway via caspase-8 and intrinsic pathway with activation on caspase-9. Both pathways resulted in cleavage on caspase-3 and 7. Among the PMFs studied, 5,7,4′-TMF was the most potent anticancer compound. In addition, crude extract and PMFs also strongly inhibit cell migration and invasion in the 50 % to 80 % range. Meanwhile, the inhibition of MMP-2 and MMP-9 was also significant, with a suppression range of 10 to 30 %. However, most current studies focus on crude extract only, and the potential use of PMFs as a chemopreventive agent was not thoroughly studied. Nonetheless, the review suggests that both crude extract and PMFs metabolites demonstrate anticancer properties and may reduce cell progression and metastasis.

6.1 In vitro anti-obesity activity of K. parviflora and its PMFs

Inhibition of α-amylase and α-glucosidase enzymes delays the breakdown of polysaccharides through competitive binding at the enzymatic site, which helps to alleviate postprandial blood glucose levels (Lebovitz, 1997). Treatment of PMFs isolated from ethanolic KPE demonstrates potent inhibitions on the α-glucosidase enzyme (Azuma et al., 2011). Compound 3,7,3′4′-TeMF and 5,7,4′-TMF significantly inhibit the enzyme (IC₅₀ = 20.4 and 54.3 µM), higher than PMF's quercetin (59.5 µM). The position of the methoxy group greatly influenced the binding affinity of the PMFs, as discussed in the previous sections (2.). In another study, the crude extract significantly inhibits α-amylase and α-glucosidase enzymes with IC₅₀ values of 433.3 μg/ml and 3.722 μg/ml. However, the inhibitory effect was weak compared to the well-known α-glucosidase inhibitors, acarbose, with IC₅₀ values of 5.10 μg/ml and 0.06 μg/ml, respectively (Yagi et al., 2019).

6.1.1

6.1.1 Reduction of triglycerides (TG) by lipolysis

Obesity is linked to increased hypertrophy of adipocytes due to the stimulation of inflammatory mediators by macrophage infiltration that mediates adipokines secretion and insulin-sensitizing signaling in adipocytes, elevating insulin resistance (Okabe et al., 2014); meanwhile, high-calorie intake suppressed lipolysis, which reduced the hydrolysis of triglycerides. The combined effect of these factors results in lower adipocyte differentiation and reduced storage of synthesized triglycerides. Excessive triglycerides accumulated in adipocyte tissue cause adipocyte hypertrophy, which leads to obesity (Schweiger et al., 2006; Ye and Gimble, 2011). KPE and its PMFs significantly upregulate genes and protein expression of mature adipocytes and reduce TG accumulation via lipolysis (Fig. 2) in 3 T3-L1 cells (Okabe et al., 2014). Treatment of 3 and 10 µg/ml crude extract significantly stimulates adiponectin mRNA expression and enzyme lipases (ATGL & HSL) in a concentration-dependent manner. At higher concentrations (10 to 30 µg/ml), isolated PMFs significantly reduced the accumulation of TG with 5,7,4′-TMF, demonstrating the most potent effect (0.3 mg/mg) compared to the vehicle control (0.7 mg/mg). High glycerol levels proved that hydrolysis of TG takes place.

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Fig. 2

The mechanism of anti-obesity and antidiabetes effect of reported PMFs and KPE.

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6.2 In-vivo anti-obesity activity of KPE and its PMFs

Antidiabetic studies on TSOD mice were conducted by Kobayashi et al. (2016) on brown adipose tissues. At both concentrations (0.3 % & 1 %), KPE significantly reduced the brown adipose tissue weight dose-dependently. The mRNA expression of UCP-1 and its marker, β3AR, was upregulated at a higher concentration than the control. The result was congruent with the previous study, with the treatment of 1 % crude extract significantly elevated UCP-1 expression to more than 200 % compared to the control (Yoshino et al., 2014). Both concentrations of KPE (0.5 & 1.0 %) potently reduced abdominal fat weight and TG buildup. At 0.5 % crude extract, the neurotransmitters (noradrenaline and adrenaline) concentration was significantly elevated compared to the control. Neurotransmitters are responsible for increased cyclic adenosine monophosphate (cAMP) levels that regulate UCP-1 mRNA expression in brown adipocytes through the cAMP/PKA pathway (Chen et al., 2013; Koh et al., 2007). In another study, KPE reduced the adipose tissue weight and plasma glucose level and elevated the release of adiponectin and stronger pparγ activity, contributing to lower insulin resistance, adipocyte differentiation, and fat accumulation (Ochiai et al., 2019). In an 8-week study on similar mice models, the growth of visceral and subcutaneous fats was suppressed moderately (Akase et al., 2011). Crude extract at both concentrations (1 % & 3 % KPE) significantly reduced the risk of dyslipidemia in TSOD mice, with potent inhibition was observed when treated with 3 % crude extract. Among the parameters, insulin and TG levels were the most suppressed by crude extract to 2.9 and 166.0 mg/dL, compared to the vehicle control 10.3 and 234 mg/dL, respectively. On another note, the crude extract also reduced the blood pressure and glucose levels of TSOD mice. At the same time, no significant change was recorded in Tsumura Suzuki Non-Obesity (TSNO) mice, proving that the crude extract did not affect the healthy or non-diseased group.

In another study, a low dosage of KPEs reduced the metabolic risk in TSOD and TSNO mice (Shimada et al., 2011). On week 8, 1 % crude extract potently reduced abdominal fat in the TSOD group. In addition, 1 % crude extract concentration decreased the blood glucose level to less than 400 mg/dL compared to the control and 0.3 % crude extract in the TSOD group. At low concentrations (0.3 %), the blood glucose level decreased after 1-hour treatment. One of the possible pathways to reduce the risk of obesity is inhibiting the lipase enzyme's activity (Heck et al., 2000). The crude extract, 5-Hydroxy-3,7-Dimethoxyflavone (5H-3,7-DMF), 5H-7,4′-DMF, and 5H-7-MF potently inhibit lipase enzyme activity compared to other PMFs with IC₅₀ values of 487, 291, 220, and 291 µg/ml, respectively. In a study by Hidaka et al. (2017), three different extracts of K. parviflora; crude KPE (KPE), PMFs-rich extract (PMF), and PMFs-poor extract (KPX), were investigated for anti-obesity effect. The result shows that the PMFs group improved the expression of the pparγ gene, which is involved in adipocyte differentiation. Meanwhile, a similar result was obtained from the previous study, with a reduction in visceral and subcutaneous fat for all three KPEs. The result was contributed by the downregulation of subcutaneous fat thickness, with the size of adipocytes decreasing to smaller cells.

8.1 Inhibition on Ache

Inhibition of acetylcholinesterase (AChe) and butyrylcholinesterase (BChe) enzymes has been a therapeutic target to alleviate Alzheimer’s disease through inhibition of hydrolysis of AChe (Nordberg et al., 2013; Rees & Brimijoin, 2003). Among the isolated PMFs, treatment of 0.1 mg/ml of compound 5,7,4′-TMF and 5,7-DMF was the most potent in suppressing both AChe and BChe enzymes, respectively (Sawasdee et al., 2009). The percentages of inhibition by 5,7,4′-TMF on each respective enzyme were 47.1 % and 46.2 %; meanwhile, 5,7-DMF were 42.6 & 84.6 %, respectively. Significant inhibition of both enzymes by PMFs contributes to another in-depth study by Seo et al. (2017) to investigate the anti-acetylcholinesterase activity of 5,7-DMF, 5,7,4′-TMF, and PeMF. Interestingly, PeMF demonstrates the most potent inhibitory effect at a concentration of 40 µM with a reduction of up to 80 % activity in contrast to 5,7,4′-TMF and 5,7-DMF (20 %) when treated at high concentration (100 µM). However, the potent activity of PMFs was insignificant due to the high neurotoxicity recorded against PC12 cells.

Therefore, PC12 cell lines were extensively used in an in vitro Alzheimer’s disease study to investigate the target compounds' neurotoxicity and neuron damage effect (Tong et al., 2018). Meanwhile, no significant cell damage and neurite outgrowth were observed for 5,7,4′-TMF and 5,7-DMF, which makes it the potent Ache inhibitor.

8.2 Increase neurite outgrowth

Interference of CRE-dependent transcription has been conjectured to promote neuronal dysfunction and induced death. Furthermore, the cAMP-stimulated signaling pathway inactivation mediates the neurite outgrowth through protein kinases (ERK/MAP) associated with cognitive impairment (Natsume et al., 2020). Isolated PMFs from KPE show intense activity to facilitate activation of the CRE-mediated transcription to ameliorate memory loss (Natsume et al., 2020). KPE and 4 PMFs compounds (30 μg/ml) demonstrated upregulation of CRE-mediated transcription by 3 to 9-fold through firefly luciferase assay. On another note, all PMFs and the crude extract do not exhibit significant toxicity against PC12D cells, a new subline from normal cells PC12D, including neurite outgrowth response from cAMP (Katoh-Semba et al., 1989), compared to the previous study.

8.3 Inhibition of beta-secretase 1 (BACE1) activity

The progression of the neurodegenerative disorder is accelerated by the growth of amyloid plaque, neurotoxic β-amyloid (Aβ) (Hardy & Allsop, 1991). Formation of Aβ peptides as a result of enzymatic cleavage of amino acid position 1 on the Aβ precursor protein by BACE1 (Vassar & Kandalepas, 2011). Inhibition of BACE1 reduced the generation of Aβ peptides responsible for elevated levels of cognitive impairment, neuronal damage, oxidative stress, and inflammatory activity (Fukumoto et al., 2010). Aβ peptides, together with the formation of neurofibrillary thread with neurons by Tau proteins, are responsible for alleviating cognitive dysfunction (Tong et al., 2005). PMFs isolated from KPE significantly inhibit the activity of the BACE1 enzyme in a dose-controlled manner. Compounds 5,7,4′-TMF demonstrate the most potent inhibitory activity with 80 % secretion reduction of BACE1 enzyme and IC₅₀ value of 36.9 µM. Meanwhile, 5,7-DMF and PeMF display significant suppression on BACE1 (49.5 µM & 59.8 µM), respectively (Youn et al., 2016). Nevertheless, all compounds exhibit higher activities than polyphenol resveratrol, a notable natural compound with therapeutic potential in Alzheimer's disease (Jabir et al., 2018). Furthermore, the molecular docking simulation portrayed the binding orientation of the PMFs on the allosteric site of the BACE1 enzyme, demonstrating the non-competitive binding inhibition by the PMFs.

8.4 Inhibition of neurotransmitter glutamate toxicity

Oxidative stress injury is a leading pathway of glutamate neurotoxicity (Lau & Tymianski, 2010). Excessive levels of glutamate neurotransmitters are correlated with overexpression of oxidative stress and damage by ROS and RNS that upregulate protein kinases and transcription factors, thus inducing apoptotic HT-22 cell death (Xin et al., 2019). The antioxidant properties of K. parviflora rhizome had a therapeutic potential to reduce toxicity induced by neurotransmitter glutamate in neuronal HT-22 cells. KPE significantly reduced glutamate-induced toxicity in HT-22 cells in a dose-controlled manner (Tonsomboon et al., 2021). Although 5 μg/ml of the crude extract could not reverse the toxicity effect, higher concentrations of crude extract (50 & 75 μg/ml) significantly ameliorated the glutamate-induced toxicity, with cell viability increasing to 115.97 % and 102.11 %, respectively. The elevated level of glutamate-induced toxicity of more than 100 % due to the upregulation of ROS expression was significantly inhibited by KPE at 50 and 75 μg/ml concentrations. Both concentrations of crude extract reduced the ROS level by 26.44 % and 46.37 %, respectively. The crude extract demonstrates antiapoptotic roles on HT22 cells by reducing cell death to 22.49 % and 28.00 %, respectively. The antiapoptotic pathway shown by KPE was revealed with suppression of MAPK pathway and ERK phosphorylation, along with upregulation of brain-derived neurotrophic factor, BNDF expression, indicating the KPE displays neuroprotective effect against glutamate-induced toxicity in neuronal HT022 cells.

8.5 Ameliorates VPA-induced cognitive impairments

Meanwhile, an in vivo study was conducted on Male Sprague Dawley rats by Welbat et al. (2016) to investigate the protective effect of KPE against drug-stimulated spatial memory impairment, valproic acid (VPA). VPA is an anti-epileptic drug for the treatment of bipolar and schizophrenia disorder, which is categorized with a high safety profile. However, several studies have reported its side effect, which includes mild memory impairment (Hommet et al., 2007) and lower hippocampal neurogenesis (Umka et al., 2010). Synergistically, combine treatment of KPEs and VPA significantly reversed the spatial cognitive impairment with higher mean preferential index (PI) in object exploratory tests on the rats. In addition, the crude extract did not show any toxicity indicator against Ki-67 cells; a higher cell number was recorded when treated with KPE compared to VPA treatment alone. Furthermore, administering VPA significantly suppressed the expression of BNDF and doublecortin proteins, a neurogenesis marker. Co-treatments of the crude extract with VPA potently reversed the effect and upregulated the expression of BNDF and doublecortin proteins up to 100 % activity.

As discussed in the previous section (8.4.), elevated free radicals are associated with oxidative damage to the cells, affecting cognitive and psychological function. Therefore, inhibiting oxidative stress by introducing antioxidant agents could provide an alternative mechanism to prevent neurodegenerative effects (Gilgun-Sherki et al., 2001). Consumption of 150 to 250 mg/kg of KPE shows neuropharmacological activities in rats’ model in 2 2-week study with antidepressant behavior and moderate enhancement of cognitive function (Hawiset et al., 2011). Meanwhile, introducing 200 mg/kg KPE significantly ameliorated the duration of escape latency of rats’ models in 7,14, and 21 days studied, compared to the vehicle-stress models (Wattanathorn et al., 2013). Nonetheless, 100 mg/kg and 300 mg/kg crude extract did not influence changes compared to the vehicle-stress models. On another note, in similar doses, the treated rats demonstrated the development of cholinergic neurons excitatory; however, the effect was only observed on neurons C3. Nonetheless, the results have shown that the properties of K. parviflora improved cognitive deterioration, as supported by the previous study at 200 mg/kg crude extract doses (Phachonpai et al., 2012).

11.1 Antifungal

The crude KPE demonstrates antifungal activity by inhibiting dermatophyte fungi activity. Moderate antifungal effect of crude extract (200 µg/ml) was recorded on T. rubrum, T. mentagrophytes, and M. gypseum with minimum inhibitory concentration (MIC) values of 62.5, 125, and 250 µg/ml, respectively. The result demonstrates that all PMFs had a weaker antifungal activity, with the highest recorded by 3,5,7-TMF (MIC value: 250 μg/ml) (Kummee et al., 2008). The low antifungal activity of PMFs contradicted the previous study by Yenjai et al. (2004), which reported PMFs 3,5,7,4′-TeMF and 5,7,4′-TMF exhibit potent antifungal activity on Candida albicans with IC₅₀ value of 39.71 and 17.63 μg/ml. In addition, both compounds possessed moderate antimycobacterial activity (MIC: 200 & 50 μg/ml) compared to the antifungal drug. The moderate antifungal activity of PMFs was further supported by the recent antifungal activity of methoxyflavones derivatives isolated from the roots of Deguelia duckeana. The isolated 7-Methoxyflavones derivatives demonstrate weak antifungal activity on C. albicans, C. gattii, and C. neoformans, with all MIC₅₀ recorded exceeding 320 µg/ml. However, isolated chalcone derivatives, 4-Hydroxylonchocarpine, from the same crude extract demonstrated potent MIC₅₀ with the lowest value recorded on C. neoformans (20 µg/ml). The result suggests that different bioactive PMFs compounds may result in distinct antifungal activities that require further studies.

11.2 Antibacterial activity

One of the earliest antibacterial studies of K. parviflora extract by Kummee et al. (2008) demonstrated negligible activity against gram-positive bacteria (S. aureus, S. epidermidis & E. faecalis) and gram-negative bacteria (E. coli). Among the genus Kaempferia studied, K. parviflora shows the most promising antibacterial activity against bacteria K. pneumoniae, P. aeruginosa, and A. baumannii, with MIC values of 64, 64, and 32 μg/ml, respectively (Sookkhee et al., 2022). Meanwhile, the crude extract also inhibits bacterial growth of Cronobacter spp and Enterohemorrhagic E. coli in a concentration-controlled manner (Jeong et al., 2016). In another study, the crude extract demonstrated potent antibacterial on P. acnes and S. Aureus with 100 % suppression on both bacteria growth when treated at a concentration of 250 and 500 μg/ml, respectively (Jin and Lee, 2018).

The influence of solvent extraction on the antibacterial activity of K. parviflora was elucidated by Sitthichai et al. (2022). Ethyl acetate extract demonstrates the most potent activity on bacteria S. epidermis with an MIC value of 3.84 mg/ml. Meanwhile, acetone, ethyl acetate, and dichloromethane-crude extract show potent inhibition on bacteria growth of C. acnes with an MIC value of 0.03 mg/ml. However, no significant difference in antibacterial activity was reported for S. aureus when treated with all crude extract. In another study, a similar result was obtained with ethyl acetate crude extract exhibiting the highest inhibition growth against H. pylori (MIC = 32 μg/ml), followed by hexane (64 μg/ml), methanol (64 μg/ml) and volatile oil (1024 μg/ml), respectively (Chaichanawongsaroj et al., 2010). Extraction of plant extract with ethyl acetate solvent to achieve maximum antimicrobial activity was also supported by another study on seeds of Peganum harmala L by (Arif et al., 2022). The ethyl acetate extract demonstrates the highest activity against S. aureus (81.10 %) and E. coli (61.29 %). Nevertheless, in a similar study on stem extract, n-butanol is the best solvent against K. pneumonia (32.44 %) and E. coli (37.61 %).

The crude extract was purified and isolated to obtain PMFs. Compounds 3,5,7-TMF, 5,7,4′-TMF, and PeMF demonstrate the most potent antibacterial activity with MIC value range between 50 and 100 μg/ml. However, the activity of the PMFs was unsatisfactory compared to the positive control, Gentamicin. Nevertheless, the combination of antibiotic Gentamicin and the PMFs produced a synergistic effect, with compound 5,7,4′-TMF recording the highest median fractional inhibitory concentration index (FICI) value (FICI value range: 0.4–0.5), compared to 3,5,7-TMF and PeMF alone. Interestingly, the combination therapies result in more potent inhibition with lower IC₅₀ than single Gentamicin treatment alone. The mechanisms responsible for the effect were not elucidated. However, it may be attributed to the anti-efflux activity, which had been reported by various metabolites from plant extracts against gram-negative bacteria (Soto, 2013). The positive results indicate potential combined uses of isolated herbal KPE against emerging Carbapenem-resistant bacteria (Iovleva & Doi, 2017).

11.3 Antiviral activity

The antiviral activity of KPE from water and ethanol was investigated against the H5N1 virus (Sornpet et al., 2017). The solvent crude extract significantly inhibits the virus replication in the first 24 h post-infection and 100 % inhibition upon 72 h infection. The potent antiviral effect of the crude extract contributed by the upregulation of cytokines TNF-α and Interferon beta-1b (IFN-β) expression in the MDCK cells on 24 and 48-hour post infections. Both cytokines are well-known for their roles as a defense mechanism in reducing disease risks by the hepatitis C virus (Mochizuki et al., 2004) and the human influenza virus (Seo & Webster, 2002). In addition, the antiviral activity of K. parviflora methanolic extract against HIV-1, Hepatitis C, and human cytomegalovirus shows promising activity (Sookkongwaree et al., 2006). K. parviflora methanolic extract shows higher activity and moderately inhibits the respective virus’s replication by 37.1 %, 17.7 %, and 29.6 %, respectively. In addition, higher concentration crude extract (200 μg/ml) significantly elevated the virus protease inhibition to 81.5 %, 71.9 %, and 74.7 %, respectively. Due to the remarkable activity of the KPE, PMFs were isolated from the crude extract to investigate the potent compounds responsible for the antiviral effect. Compound 5H-7-MF and 5,7-DMF show potent antiviral activity against the HIV-1 protease virus with IC₅₀ values of 19.04 and 19.54 µM. Nonetheless, PMFs show weak inhibition with negligible IC₅₀ value against hepatitis C and human cytomegalovirus. Nevertheless, flavone groups are well-known for their antiviral activity through multiple mechanisms of action. Compound quercetin demonstrates potent activity by mediating the Internal Ribosome Entry Sites (IRES) translation and reducing HSP40 and HSP70 interaction with NS5A (Gonzalez et al., 2009). Meanwhile, a similar compound approached a different mechanism by suppressing the activity and replication of HCV NS3 protease (Bachmetov et al., 2012). Apart from that, inhibition of both viruses' reverse transcriptase and DNA polymerase enzyme and suppressing nucleoprotein production are other possible pathways to achieve antiviral activity of flavones, a flavonoid subclass group (Ben-Shabat et al., 2020).

11.4 Antiparasitic activity

In addition, inhibition of parasitic growth is crucial to preserving human health. Parasites Plasmodium spp. was responsible for the widespread malaria disease, anemia, and acidosis (Phillips et al., 2017). A study by Yenjai et al. (2004) showed that the isolated PMFs, 5,7,4′-TMF and 5,7,3′,4′-TeMF demonstrate potent suppression on Plasmodium falciparum (IC₅₀: 3.70 and 4.06 μg/ml). On another note, the infectious parasite Toxoplasma gondii may cause miscarriages and neurological issues (Montoya & Liesenfeld, 2004). A recent study depicts that KPEs can suppress parasite growth of Plasmodium and T. gondii with IC₅₀ values of 28.7 μg/ml and 53.5 μg/ml, respectively (Leesombun et al., 2019). The crude extract's inhibitory effect on T. gondii was significantly more potent than the commercialized sulfadiazine, indicating that an in-depth study on the antiparasitic effect of KPE may provide an alternative treatment for toxoplasmosis.

13.1 Structural modification of PMFs isolated from KPE

A study by Yenjai et al. (2009) hypothesized that structural modification of PMFs isolated from the crude extract might improve the polarity and solubility. Compound 5,7-DMF was modified through a series of reactions to produce flavanones, dihydroxyflavone, chalcone, and oximes derivatives. In anticancer analysis, derived compound 5,7-Dihydroxyflavone shows potent cytotoxicity against human liver cancer cell (HepG2) and breast cancer cell lines (T47D), with IC₅₀ value of 21.36 and 25.00 μg/ml, respectively. However, in antifungal activity, only 5,7-Dihydroxyflavanone oxime demonstrates potent activity (IC₅₀: 48.98 μg/ml); meanwhile, for antimalarial activity, compound 5,7-DMF exhibits potent inhibitory (IC₅₀: 5.73 μg/ml), comparable with the standard drug, Dihydroartemisinin (IC₅₀: 5.73 μg/ml). In another study, compound 5,7-DMF was modified to incorporate the amino and nitro derivatives for anticancer activity against the human epithelial carcinoma cancer KB cell line (Wanich & Yenjai, 2009). The modified compounds, chalcone and amino flavones, induced potent cytotoxicity against the cancer cell with IC₅₀ values of 6.80 and 5.84 μg/ml, respectively. Likewise, in another study, oxime derivatives from 5,7-DMF show potent inhibition against KB and NCI-H187 cancer cell lines with IC₅₀ values of 0.26 and 0.014 μg/ml, respectively, more potent than the Ellipticine control agent (IC₅₀: 1.79 & 2.77 μg/ml) (Yenjai & Wanich, 2010). In another study, a combination of selenium nanoparticles (SeNP) and KPE shows potent cytotoxicity toward gastric cancer cells (AGSr) (Wang et al., 2022). The new approach was introduced as a previous study demonstrated the SeNP potent’s bioactivities in medical applications due to its low toxicity, biocompatibility, and high bioavailability properties (Bisht et al., 2022). Based on the study, treatment of 200 μg/ml complexes significantly induced cytotoxicity in a dose-dependent manner against AGS gastric cancer cell line with more than 70 % reduction in cancer cell growth. Interestingly, the KP-SeNP complex exhibits high selectivity in normal cells. The normal cell growth remained above 80 %, compared to 50 µM cisplatin, which inhibits cell viability by 70 %. The complex induced apoptotic cell death via inhibition of PI3K/AKT/mTOR phosphorylation, which activates the BCL-2 pro-apoptotic protein and caspase 3, resulting in cell death. The result suggests modification of PMFs from KPE capable of elevating cytotoxicity against cancer cells and reducing its lipophilicity character.

13.2 Transdermal delivery mechanism of KPE

In addition, the bioavailability of KPE was potently enhanced through the combination of transdermal vehicles isopropyl myristate (IPM), ethanol, and propylene glycol, with the addition of permeation enhancers and fatty acids. The solubility of K. parviflora in the IPM vehicle was recorded as 15.6 mg/ml and significantly improved when combined with a higher ethanol-to-propylene glycol ratio. Interestingly, the combination of ethanol and IPM vehicle in all proportions (1:9, 2:8 & 3:7) significantly improved the solubility to 94.1, 182.4, and 204.4 mg/ml, respectively. A higher ethanol ratio shows significant enhancement in crude extract solubility. Nonetheless, it reduced the flux of the crude extract. Ethanol with a low IPM ratio (1:9) demonstrates the highest flux in the crude section (314.7 μg/cm²/h). Higher flux value indicates improvement in solubility properties of KPE in ethanol: IPM vehicles (Tuntiyasawasdikul et al., 2014). On another note, adding fatty acid and oleic acid with ethanol: IPM vehicle significantly improved the solubility and flux of KPE to 109.8 mg/ml and 358.5 μg/cm²/h, respectively. The study suggests that adding fatty acid could reduce the resistance of the outer layer of skin during transdermal transport to improve its effectiveness. A similar study was designed by Tuntiyasawasdikul et al. (2015) to investigate the permeability of a monolithic drug-in-adhesive type patch of isolated PMFs from crude KPE with a combination of the delivery vehicle and permeation enhancer, oleic acid, and methanol. Combining oleic acid and methanol produced maximum flux values for all compounds, indicating a synergistic effect between K. parviflora, oleic acid, and methanol content. Thus, an in vivo study on rats with patch combinations significantly released the PMFs into the systemic circulation through transdermal delivery with plasma drug concentration and time maximum was recorded at 218.08 ng/ml and 8 h, respectively, with less than 1 % retained in the skin (1.43 μg/cm²). Based on these results, microemulsion and microgels of KPE were introduced through combination with oleic acid and surfactants, which has been shown to improve the crude extract solubility and dissolution rate in transdermal delivery mechanism (Rangsimawong et al., 2018). The microemulsion of KPE improved the skin permeation in a time and dose-dependent manner.

Interestingly, the addition of Limonene at a higher ratio (10 %) improved the permeation capacity and recorded the highest flux value (0.132 μg/cm²/h) compared to the control (0.013 (μg/cm²/h). The result was consistent with the study by (Lu et al., 2014). Limonene is a nonpolar compound with a lipophilic character that facilitates drug diffusion into the skin. Meanwhile, microgels of KPE produced similar results with higher permeability than the control. However, the permeation flux was lower than the microemulsion state, with 0.044 μg/cm²/h recorded. Microgels are associated with high viscosity, leading to lower drug diffusion to penetrate the skin. Thus, the permeation and flux rates were drastically reduced (Gannu et al., 2010).

13.3 Self-emulsifying delivery system of KPE

The self-microemulsifying delivery system (SMEDDS) was introduced on KPE by Mekjaruskul et al. (2013). Previous study has demonstrated that combining the crude extract with oil and surfactants improved the solubility and permeability of drug delivery. SMEDDS is a microemulsifying process through chemical interaction contrary to mechanical methods prepared by the previous study to form a homogenous mixture of crude KPE (Kim et al., 2019). In an in vitro study, the combination of KPE with polyethylene castor oil and propylene glycol in a ratio of 2:1 (80 & 85 %) with triglycerides of coconut oil (20 & 15 %) improved the dissolution rate compared to the crude extract with 100 % unloaded within 20 min. In addition, the crude extract combined with the cyclodextrin complex demonstrates a higher dissolution rate of 100 % within 10 min. Meanwhile, for the in vivo study, a similar result was obtained, with all complexes showing enhancement in oral bioavailability compared to crude extract. In a separate study, a self-nanoemulsifying delivery system (SNEDDS) significantly improved the dissolution rate of crude extract. However, it recorded lower capacity than SMEDDS, with 92 % unloading after 60 min. The significant differences may be attributed to the type and ratio of oil, surfactant, and co-surfactant used (Chairuk et al., 2020). However, the formation of nanosuspension crude extract in liquid form improved the dissolution rate to over 80 % in both 3 % SLS and PBS stabilizer at pH 6.8, comparable with the SMEDDS. Meanwhile, in solid form, both stabilizers slow down the dissolution rate in the first 30 min before achieving a similar rate as a liquid form within 1 h. The result indicates that micro and nano-drug delivery may improve the lipophilicity character and bioavailability of the PMFs from KPE (Mekjaruskul and Sripanidkulchai, 2020).

13.4 Improvement of solubility and dissolution rate of KPE and its PMFs

In another study, introducing complex Hydroxypropyl-β-cyclodextrin (HPβ-CD) and 5,7-DMF from KPE significantly improved the solubility compared to free 5,7-DMF from 0.00304 to 1.10 mg/ml (Songngam et al., 2014). The complex was investigated on BChE inhibitory, as a previous study reported 85 % inhibition on the enzyme by 5,7-DMF (Sawasdee et al., 2009). The HPβ-CD/5,7-DMF complex significantly inhibits the enzyme activity in a concentration-dependent manner with an IC₅₀ value of 23.5 µM, 2.7-fold stronger than free 5,7-DMF (IC₅₀: 62.4 µM). HPβ-CD has been widely used to improve the solubility of flavonoid metabolites due to its low toxicity and high compatibility (Loftsson and Duchêne, 2007). The solid dispersion method was introduced to enhance the dissolution rate of PMFs with a combination of hydroxypropyl methylcellulose (HPMC) and polyvinyl alcohol-polyethylene glycol grafted copolymer (PVA-co-PEG) polymer (Weerapol et al., 2017). In the solid dispersion technique, the hydrophobic drug (5,7,4′-TMF) was mixed with a hydrophilic matrix (polymer) to form a homogenous mixture of amorphous or crystalline particles (Paudel et al., 2013). In the absence of a polymer matrix, there was no significant difference in the dissolution rate of 5,7,4′-TMF dissolved within 120 min of exposure. Interestingly, the formation of 5,7,4′-TMF and HPMC with a ratio of 1:2 demonstrate the dissolution rate of 43.71 % and 62.11 %, respectively. Interestingly, the mixture with polymer PVA-co-PEG shows the highest dissolution rate compared to the HPMC polymer combination. PVA-co-PEG ratio (1:1) enhanced the solubility rate to 75.74 % and 84.83 %, respectively. However, further increases in polymer ratios with the compound 5,7,4′-TMF (4:1) significantly reduced the dissolution rate. Compared to the isolated compound from KPE, the excess polymer ratio causes increased mixtures' viscosity, thus reducing the dissolution rate (Weerapol et al., 2017).