Phytochemical Screening and High-performance Thin-layer Chromatography Quantification of Vitex trifolia Leaves Hydro-alcoholic Extract: Potential Anti-inflammatory Properties

Aims: The present study aims to evaluate the phytochemical composition of Vitex trifolia (V. trifolia) leaves hydro-alcoholic extract and to report for the first time, its phenolic content using a validated high-performance thin-layer chromatography (HPTLC) method. Study Design: Qualitative phytochemical analysis and HTLC densitometric quantitative analysis. Place and Duration of Study: The study was carried out at the Faculty of Pharmacy, Universiti Teknologi MARA (UiTM), Malaysia, from March 2020 to December 2020. Methodology: The preliminary phytochemical screening was carried out qualitatively. The HPTLC analysis was performed on glass-backed 60 F254 silica gel plates using a two steps gradient elution Original Research Article Ghafari et al.; JPRI, 33(28A): 111-121, 2021; Article no.JPRI.68297 112 method of the mobile phase. In the first step, methanol was used to develop the plates until 40 mm of developing distance, while in the second step, plates were developed with n-hexane:ethyl acetate:acetic acid (20:9:1, v/v/v) until 80 mm of developing distance. Detection and quantification were performed by densitometric analysis at 254 nm. The method was validated as per the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guideline in terms of linearity, precision, accuracy, the limit of detection (LOD), and the limit of quantification (LOQ). Results: The preliminary phytochemical screening of V. trifolia leaves hydro-alcoholic extract showed the presence of alkaloids, flavonoids, phenols, phytosterols, and terpenoids. The developed HPTLC method was proved to be linear, precise, and accurate. The LOD and LOQ of the method were determined to be 2.01 μg/band and 6.08 μg/band, respectively. The total phenolic content of the extract was calculated from the standard gallic acid calibration plot and found to be 136.94 ± 4.02 mg gallic acid equivalent (GAE)/g of dried extract. Conclusion: This preliminary study revealed that V. trifolia has a considerable amount of phenolic compounds, which can potentially contribute to its anti-inflammatory, antioxidant, and anticancer activities. Further pharmacological investigations are being carried out to support the folkloric claims.


INTRODUCTION
In recent years, the pharmaceutical application of plant-based natural products has gained interest. Reports from the World Health Organization (WHO) indicate that near 65% of the world's population use plants derived natural products for their health care. Most of these products are used accompanied by conventional drugs. Approximately around 150000 plant species have been evaluated so far and many of them have been reported to possess therapeutic properties. The therapeutic effects of them are due to the presence of different secondary metabolites. However, exploring their complex chemical compositions has always been challenging for researchers [1].
Vitex trifolia (V.trifolia), a species from the family Verbenaceae, is a multi-purpose medicinal plant that is widely distributed in tropic and sub-tropic regions including Malaysia, Thailand, Indonesia, Australia, India, and Sri Lanka [2][3]. The plant is a shrub with trifoliate leaves and purple inflorescences [4][5]. It is traditionally used to treat inflammation, pain, fever, and allergy [3,6]. V. trifolia's infusion and decoction are used to treat intestinal complications, tuberculosis, and amenorrhea [2]. It is also used as an emmenagogue as it stimulates the blood flow in the pelvic. Moreover, In Unani medicine, the plant is known as sambhalu and it is used to increase libido. The inner bark of the plant is useful for the treatment of diarrhoea, cough, hypertension, and sinusitis while the root is used as anthelmintic and diuretic medicines [7]. The fruits and leaves are used to treat inflammation and the flowers are useful to treat fever. The antiinflammatory and antioxidant effects of V. trifolia were reported by several studies [8][9][10]. The plant can exhibit its anti-inflammatory effect by suppressing the expression of pro-inflammatory mediators such as cytokines, cyclooxygenase, and inducible nitric oxide synthase through modulation of inflammation-related signalling pathways including, nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) signalling pathways [9,[11][12]. On the other hand, the plant's free radical scavenging ability is responsible for its antioxidant activity [2,13]. Additionally, V. trifolia can also possess antibacterial [14][15], antiviral [16][17], anticancer [18][19], hepatoprotective [20], antiasthmatic [7], antihistaminic [21][22], anti-diabetic [23], and anthelminthic [24] properties.
The multi-pharmacological effects of V. trifolia are due to the presence of several secondary metabolites including polyphenols, terpenoids, glycoside [25], alkaloids, phytosterol [26], and iridoids [27]. Polyphenolic compounds are playing a major role in the plant's antiinflammatory, antioxidant, and anticancer activities [28]. Several studies reported the isolation of several polyphenolic compounds from V. trifolia. Casticin or vitexicarpin is one of the main polyphenolic compounds of genus Vitex including V. trifolia and reports suggest that it can display potent anti-inflammatory and anticancer properties [10,12]. Additionally, artemetin, chrysosplenol D, luteolin, penduletin, persicogenin, quercetin, vitexin, and isovitexin are among polyphenolic compounds that have been isolated from the leaves, fruits, and aerial parts of the plant [7,[28][29][30][31] However, as per our knowledge from the literature, so far there is no report about the quantification of total phenolic content (TPC) of V. trifolia leaves hydro-alcoholic extract which is important for further pharmacological investigations. Thus, this study was aimed to evaluate the chemical composition of V. trifolia leaves hydro-alcoholic extract and to report for the first time, its TPC using a validated highperformance thin-layer chromatography (HPTLC) method.

Sample Extraction
V. trifolia leaves were collected from Subang Jaya, Selangor Darul Ehsan, Malaysia, and the plant was authenticated and deposited at the Atta-ur-Rahman Institute for Natural Product Discovery (AuRINs), Universiti Teknologi MARA, Puncak Alam, Selangor Darul Ehsan, Malaysia with a voucher specimen no. 0215USJ. Clean leaves underwent shed drying for 5 days and grounded into a fine powder using an electrical blender. The powdered leaves (150 g) were macerated in ethanol and water (1:1, v/v) under sonication for 30 min at room temperature (23-26 o C) [32]. Next, a rotary evaporator was used to remove the solvent from the sample at 40 o C and 100 mbar of pressure, and the sample was then freeze-dried at -80 o C. The final extract was stored at -20 o C in a light protective container.

Qualitative Phytochemical Screening
The qualitative phytochemical screening for the presence or absence of alkaloids, flavonoids, phenols, phytosterols, and terpenoids was performed according to different qualitative methods [33][34]. The presence of alkaloids was evaluated by Dragendroff's test. 3 to 4 drops of Dragendroff's reagent (potassium bismuth iodide) were added into 4 ml of extract solution. An orange precipitate indicated the presence of alkaloids in the sample. While testing flavonoids, 2 ml of the 10% sodium hydroxide was added to 4 ml of extract solution to produce a yellow colour that indicated the presence of flavonoids. For the screening of phenols, 4 to 5 drops of 3% ferric chloride were added into 3 ml of extract's aqueous solution. The existence of phenols was indicated after a blue colour appeared. Liebermann-Burchard's test was used to test the occurrence of phytosterols. The extract (50 mg) was dissolved in 2 ml of acetic anhydride and 3 to 4 drops of sulphuric acid were added. The presence of phytosterols was displayed with the appearance of a brown colour. For testing the presence of terpenoids, 0.5 ml of chloroform and 3 to 4 drops of sulphuric acid were added into 3 ml of extract's ethanolic solution. A reddishbrown ring confirmed the presence of terpenoids in the sample.

HPTLC Analysis and Phenolic Content Quantification
The HPTLC analysis of V. trifolia leaves hydroalcoholic extract was carried out according to the method by Agatonovic-kustrin et al. [35]. Gallic acid standard and plant powder stock solutions were prepared by dissolving 10 mg of the standard compound and plant powder in 10 ml TLC grade methanol. The solutions were then centrifuged at 3000 rpm for 10 min and stored in light protective containers. Chromatography was performed using an HTPLC system (CAMAG, Muttenz, Switzerland) on HPLTC glass-backed plates (20 cm × 10 cm) pre-coated with silica gel 60 F 254 with 0.20 mm layer thickness (Merck, Germany). The HPTLC plates were pre-washed with 100% methanol and dried at 100 o C for 30 min to be activated. 20 µl of V. trifolia leaves hydro-alcoholic extract solution and 2, 4, 6, 8, and 10 µl of standard gallic acid solution were applied on TLC plates as 8 mm bands using an automatic TLC sampler 4 (CAMAG, Muttenz, Switzerland) equipped with a 25 µl HPTLC syringe (Hamilton, Bonaduz, GR, Switzerland). The bands were applied with an 8 mm distance from the bottom edge, 14 mm distance from both sides, and a minimum of 2 mm of the distance was maintained between bands. An automatic developing chamber 2 (CAMAG, Muttenz, Switzerland) was used to develop the plates in the mobile phase after the sample application. For separation, a 2 step gradient elution method of mobile phase was used. In the first step, methanol was used to develop the plates until 40 mm developing distance, while in the second step, plates were developed using n-hexane, ethyl acetate, acetic acid (20:9:1, v/v/v) until 80 mm developing distance. The developed plates were photo-documented using a TLC visualizer (CAMAG, Muttenz, Switzerland) before derivatization under UV light at 254 nm wavelength and daylight, and after derivatization under daylight. The derivatization was carried out using a Chromatogram Immersion Device (CAMAG, Muttenz, Switzerland) by dipping the plates into the ferric chloride (1%) for 1 sec with an immersion speed of 5 cm/s. After derivatization, plates were dried at 100 o C for 10 min. Quantitative analysis was done with TLC scanner 3 (CAMAG, Muttenz, Switzerland) at 254 nm. The scanning speed was 20 mm/s while the slit dimension was set at 6.00 mm × 0.4 mm. The densitograms were analysed by WinCATS version 1.4.9.2001 (CAMAG, Muttenz, Switzerland).

Method Validation
The validation of the method was performed as per the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guideline [36], and the linearity, precision, accuracy, the limit of detection (LOD), and the limit of quantification (LOQ) of the method were assessed. To determine the linearity, a calibration curve was plotted using the concentration of the standard gallic acid (µg/band) against peak area (AU) and was assessed by linear regression analysis. The precision was determined by assessing the repeatability of the method using inter-day and intra-day precision methods. The repeatability was measured by measurement of peak area (AU) of standard gallic acid at three concentrations (4, 6, and 8 µg/band) under the same condition on the same day and three consecutive days and was assessed by the relative standard deviation (% RSD). The recovery rate of the standard solution was measured to determine the accuracy of the method. Spiked samples were prepared by adding known amounts of standard gallic acid at 50, 100, and 150% of the pre-analysed analyte. The samples were analysed under the same conduction in triplicate and the recovery rate was calculated. The LOD of the method is defined as the lowest concentration of the analyte in the sample mixture which is qualitatively detectable while the LOQ is the lowest amount of the analyte in the sample that can be can be quantified. The LOD and LOQ of the standard were calculated as 3.3σ/S and 10σ/S respectively, where σ is the standard deviation and S is the standard calibration curve slope.

Preliminary Phytochemical Screening of V. trifolia's Leaves Hydro-alcoholic Extract
The preliminary phytochemical screening revealed the presence of alkaloids, flavonoids, phenols, phytosterols, and terpenoids in V. trifolia leaves hydro-alcoholic extract Table 1. The presence of phytosterols, terpenoids, flavonoids, and phenols in the ethanol extract of V. trifolia leaves was also reported by a couple of different studies [13,26]. Moreover, V. negundo [13], V. agnus-castus [37], and V. glabrata [38] leaves extracts were also reported to have various secondary metabolites. This indicates that V. trifolia is rich in secondary metabolites which may contribute to its reported multipharmacological effects.

HPTLC Method Validation
With a 2 step gradient elution method of mobile phase using 100% methanol and n acetate:acetic acid (20:9:1, v/v/v) the bands were found to be separated symmetrically without any diffuseness, which indicates the suitability of the mobile phase system for the separation of the sample. The validation values for the HPTLC method are shown in Table 2. The developed method was found to be linear in the range of 2 10 µg/band with coefficient determination ( 0.973 ± 0.011. The LOD and LOQ for gallic acid were found to be 2.01 µg/band µg/band, respectively which indicated that the developed method is quite sensitive for the simultaneous quantification of phenolic compounds. The intra-day and inter precision % RSD, shown in Table 4, was recorded less than 10% which shows th is precise. The accuracy of the method, shown in Table 4, was evaluated in terms of the method's recovery rate, and the average recovery rate was found to be 99.71% which shows good accuracy chematic diagram for extraction and phytochemical screening of V. trifolia With a 2 step gradient elution method of mobile phase using 100% methanol and n-hexane:ethyl acetate:acetic acid (20:9:1, v/v/v) the bands were found to be separated symmetrically without any diffuseness, which indicates the suitability of the mobile phase system for the separation of the sample. The validation values for the HPTLC n in Table 2. The developed method was found to be linear in the range of 2coefficient determination (R 2 ) of 0.011. The LOD and LOQ for gallic acid were found to be 2.01 µg/band and 6.08 µg/band, respectively which indicated that the developed method is quite sensitive for the simultaneous quantification of phenolic day and inter-day precision % RSD, shown in Table 4, was recorded less than 10% which shows the method is precise. The accuracy of the method, shown in Table 4, was evaluated in terms of the method's recovery rate, and the average recovery rate was found to be 99.71% which shows good accuracy of the method as it is within the range of 90 to 110%. Thus, the method was found to be linear, sensitive, precise, and accurate.

Quantification of Phenolic Content in V. trifolia Leaves Hydro Extract
The chromatograms, shown in Fig. 2

Quantification of Phenolic Content in Leaves Hydro-alcoholic
The chromatograms, shown in Fig. 2, indicate f the sample were separated symmetrically without any diffuseness. The identification of phenols in the extract was determined by comparing the retention factor (R f ) values of the sample with that of the standard.
value of standard gallic acid was found to alcoholic extract of V. trifolia wed 6 peaks and the fourth peak, with value of 0.39, was similar to the standard value which indicates the presence of phenols in the sample Table 4. The phenolic content of the plant was determined using gallic acid calibration curve (y= 0.011), Fig. 4, and presented as mg gallic acid equivalents       [13], and 81.5 [41] mg GAE/g of dried extract, respectively. Polyphenolic compounds are a group of naturally occurred secondary metabolites in plants with more than one group of phenols [42][43]. They are widely distributed in plants and they are reported to possess several pharmacological effects including, antiinflammatory [44], antioxidant, and anticancer [43] properties. Several polyphenolic compounds were isolated from V. trifolia and other Vitex genus including, casticin, artemetin, gallic acid, quercetin, penduletin, persicogenin, vitexin, and isovitexin [8,19,28,31]. These compounds are believed to be responsible for V. trifolia's antiinflammatory, antioxidant, and anticancer effects by acting on different signalling pathways related to the pathogenesis of inflammatory diseases, oxidative stress, and cancer [8,10,12,28]. These secondary metabolites inhibit the expression of inflammatory mediators such as cytokines, leukotrienes, pro-inflammatory enzymes, and chemokines through modulation of NF-κB and MAPKs pathways [9,[11][12]. Moreover, the antioxidant activity of phenols is expressed by their free radical scavenging ability [2,13]. This preliminary study indicates that V. trifolia leaves hydro-alcoholic extract can be a good source of polyphenolic compounds as we found that it has a good amount of phenolic content. However, further investigations are needed to determine the type of phenolic compounds and their specific pharmacological effects.

CONCLUSION
From the qualitative preliminary phytochemical analysis, various secondary metabolites such as alkaloids, flavonoids, phenols, phytosterols, and terpenoids were identified in V. trifolia leaves hydro-alcoholic extract. HPTLC analysis further confirmed the presence of phenols in the plant with a considerable amount. The TPC of the plant hydro-alcoholic extract was found to be more than that of several previously reported Vitex species alcoholic extracts. The presence of phenolic compounds in a high concentration, and other secondary metabolites can potentially contribute to the plant's antioxidant and anticancer activities especially anti-inflammatory attributes. More so in the COVID-19 pandemic situation that warrants more research to be carried out to boost the immune system. Thus, the plants which are rich in potential phytochemical composition should be considered for pharmaceutical and/or nutraceutical usage. On that note, further pharmacological investigations are needed to prove these claims.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
Not applicable.

ETHICAL APPROVAL
Not applicable.