The leaves of Vitex trifolia
were collected, dried and powdered and was subjected to various phytochemical screening, extraction of crude phytochemicals and estimation of its response to various enzymes like lipoxygenase, cyclooxygenase, xanthine oxidase and tyrosinase. The preliminary phytochemical screening with the various qualitativechemical tests of various leaf extracts revealed the presence of carbohydrates, flavonoids, protein and amino acids, tannins, phytosterols and saponins phytoconstituents. The phytochemical screening of the Vitex trifolia
studied presently showed the presence of alkaloids, flavonoids and Glycosides (Table 1).
Table 1: Phytochemical screenings of aqueous methanol leaf extract Vitex trifolia.
The alkaloid and flavonoid extract of Vitex trifolia
were loaded on Precoated TLC plates (60 F2 54 Merck) and developed with a solvent system of hexane, ethyl acetate and acetic acid in the ratio of 10:5:0.5. The developed plate was viewed under UV 240 nm and 360 nm (Table 2).
Table 2: Partial characterization of different phytochemical extract from the Vitex trifolialeaves by TLC.
The concentration of flavonoids in various concentration extracts of the Vitex trifolia
was determined using spectrophotometric method with aluminum chloride. The content of flavonoids was expressed in terms of quercetin equivalent. The concentration of flavonoids in plant extract from Vitex trifolia
ranged from 20.72 to 59.26 mg/g. Methanolic extract contains the flavonoid concentration. The inhibitory effects of COX-mediated TMPD oxidation activity were examined using purified COX as enzyme sources COX-2 activity was strongly inhibited by used in this study alkaloid and flavonoid extract from vitex trifolia
inhibited COX-2 the inhibition potency quite different based on the results obtained in Graph 1.
The following experiments focused on the inhibition potential of CBDA for COX-2 activity. Whereas the standard drug Celecoxib inhibited the COX2 enzyme with an IC50 of 52 nM. The results are shown in Graph 1.
The anti-inflammatory activity of the alkaloid and flavonoid extract of vitex trifolia
was evaluated by measuring the inhibition of LOX using linoleicacid as substrate. The results were reported in Graph 2 flavonid extract showed inhibition percentage above 70.22% at 20 μg/mL.
The standard n-propylgallate showed 72% inhibition at 20 μg/mL. The alkaloid and flavonoid extract of Vitex trifolia
were tested for the effects on the oxidation of DOPA by mushroom tyrosinase. With increasing the concentrations of the alkaloid and flavonoid extract of Vitex trifolia
, the diphenolase activity of mushroom tyrosinase markedly decreased concentration dependently. The values of IC50, the inhibitor concentration leading to 50% activity lost, of Vitex trifolia
were estimated to be 5, 10, 15 and 20 μg/ml, respectively (Graph 3).
The four different concentrations of Vitex trifolia
alkaloid and flavonoid extract were tested for the inhibition activity of xanthine oxidase. Vitex trifolia
extracts in different concentrations inhibited the xanthine oxidase activity. The maximum inhibition was found at 20 μg/ml concentration (Graph 4).
In many inflammatory disorders there is excessive activation of phagocytes, production of 02-, OH radicals as well as non free radicals species (H2
), which can harm severely tissues either by powerful direct oxidizing action or indirect with hydrogen peroxide and -OH radical formed from O2
- which initiates lipid peroxidation resulting in membrane destruction. Tissue damage then provokes inflammatory response by production of mediators and chemotactic factors. The reactive oxygen species are also known to activate matrix metello proteinase damage seen in various arthritic tissues (Cotran et al., 1994).
Phytochemicals from medicinal plants showing anti-inflammatory activities have the potential of filling this need because of structures are different from those of the more studied and their those of the more action may too very likely differ (Fabricant and Fanworth, 2001
and Prachayasittikul et al., 2008).
Some studies have demonstrated that flavonoid possessanti-inflammatory activities by inhibition of cyclooxygenase-2 (COX-2) expression in lipopolysaccharide (LPS)-activated RAW 264 cells or inhibiting inducible nitric oxide (iNOS) protein and mRNA expression in LPS-activated murine J774 macrophages (Hou et al., 2005)
and such activities appear to be structure dependent. COX-2 seems to be involved in many inflammatory processes. Some antioxidants inhibit the expression of COX-2 by interfering with the signalling mechanisms that regulate the COX-2 gene (Hou et al., 2005).
From the result, it is clear that caragennan induced paw oedema for the administered dose (500 mg/kg, p.o.) is comparable with reference standard indomethacine, which is a cycloxygenase inhibitor. But anti-inflammatory activity against caragennan-induced paw oedema is also shown by lipoxygenase inhibitor. Hence inhibition of caragennan-induced paw oedema by crude extract may be due to inhibitory activity of lipoxygenase enzymes (Gupta et al., 2006).
Therefore, from the present study, we can conclude that alkaloid and flavonoid extracts of Vitex trifolia
for the dose of 20 μl/ml shows anti-inflammatory activity in the early stage as well as in the late stage (up to 180 min) and after that, the effect becomes similar to that of negative control.
Lipoxygenases (LOXs) (LOX; EC 188.8.131.52) are a family of non-hemeiron-containing dioxygenases catalyzing the biosynthesis of leukotrienes. Leukotrienes function as initiators of inflammation and their inhibition is considered to be partly responsible for the anti-inflammatory activity. In the present study alkaloid and flavonoid extracts of Vitex trifolia
showed good anti-LOX activity with an IC50 value of 29.87 μg/ml (Graph 2). LOX inhibition was used to evaluate anti-inflammatory activity of a few medicinal plants used in Limousin country. Filipendula ulmaria
(Meadow sweet) recorded LOX inhibition with IC50
of 60 μg/ml and Urtica dioica
(Nettle) methanolic extract inhibited LOX with IC50 of 348 μg/ml (Trouillas et al., 2003).
In another study, eight methanolic extract out of 18 undomesticated plants of South Africa showed significant inhibition of 5-lipoxygenase (5-LOX) activity. Bidenspilosa
extract exhibited IC50 of 21.8 μg/ml and Emexaus trails
extract recorded IC50 of 81.4 μg/ml for LOX inhibition (Wang et al., 2006).
LOXs are sensitive to antioxidants as antioxidants are involved in inhibition of lipid hydroperoxide formation due to scavenging of lipidoxy orlipidperoxy-radicals. This could lead to less availability of lipid hydro peroxide substrate required for LOX catalysis (Rackova et al., 2007
and Gentallan et al., 2019).
The effects of alkaloid and flavonoid extract of Vitex trifolia
on the monophenolase and the diphenolase activities of tyrosinase were studied. The results showed that alkaloid and flavonoid extracts of Vitex trifolia
could lengthen the lag phase of the monophenolase and decrease the steady-state rate of both monophenolase and diphenolase activity. The inhibition was displayed as reversible and the inhibition type was found to be mixed type. The inhibition constants for alkaloid and flavonoid extracts of Vitex trifolia
binding with the free enzyme (E), KI, were obtained to be 0.507 and 1.543 mM, respectively, indicating that alkaloid and flavonoid extracts of Vitex trifolia
was three times as potent for inhibiting the free enzyme. However, the inhibition constants for these two inhibitors binding with the enzyme substrate complex (ES), KIS, were obtained to be 1.354 and 1.321 mM, respectively. The values are almost the same, indicating alkaloid and flavonoid extracts of Vitex trifolia
inhibited ES complex as potently as 4-cyanobenzoicacid. In addition, anthocynin extracts of Eclipta alba
inhibited the free enzyme more potently than it did the ES complex. However, the inhibitory effect of alkaloid and flavonoid extracts of Vitex trifolia
was the contrary.