Grewia hirsuta Vahl. Hydroethanolic Leaf Extract: Phytochemical and Acute Toxicological Studies

Introduction

From time immemorial plants have been extensively used as medicines and are popular in folklore medicine. Ayurveda and Siddha systems use crude drugs as raw materials for preparing medicines. The National survey indicated that ninety percent of the public is dependent on non-allopathic systems like Ayurveda, Unani and Siddha systems of medicine.¹ Plant origin medicines either crude extract or standardized herbal extracts offer an unmatched opportunity of therapeutics due to the fact that plants have complex chemical diversity.Active principles of plant origin are of high medicinal value and needs to be isolated and evaluated for safety and efficacy profiles.² The ethnic use of medicines indicates the therapeutic application of the crude version in humans. The ethnic use medicinal is an indicative of a useful medicine which may be novel and highly useful. However, one should be careful while using in the form of concentrated extracts in humans. There are chances of extract highly unsafe and toxic. Before using in humans, phytochemical studies and safety evaluation should be scientifically established.³ The crude drug of the Grewia hirstua has been used by tribes and traditional healers as nerve tonic, antidiabetic, anticancer, anti-fertility, anti-ulcer, analgesic and anti-inflammatory.^4,5 There is a need to establish the types of phytochemicals that are responsible for imparting therapeutic value of the plant. In this study, a systematic analysis of identifying and isolation of the major active principles was established by preparing appropriate extract and subjecting to advance phytochemical evaluation. Further we established the dose of the extracts to use in preclinical and clinical studies. Herbal medicines contain phytoconstituents comprising of phenolic, flavonoids, alkaloids, tannins, leucoanthocyanins, coumarins, saponins and terpenoids. These active phytochemicals are known for antioxidant activity, activation of enzymes and anticancer activity. It is well established that oxidative stress is the root cause for many metabolic disorders.^6,7

In plants, the content of active principles varies due to environmental conditions, geographical location, species, time of collection, and anatomical part of the plant.⁸ The complexity of active principles pose the challenge of reproducibility in therapeutic activity and phytochemical similarly. This problem is addressed by standardization of herbal extract in which a unique phytochemical is used as a biomarker unlike synthetic drugs. The development of herbal medicines is beyond modern approaches like Pharmacokinetics and Pharmacodynamics (PKPD) modeling and Absorption, Distribution, Metabolism and Excretion (ADME) studies.⁹ It is mainly based on the measurement of biological activity and biomarker quantitative estimation for the administration of drugs.

If one generalizes that phytochemicals being natural products are devoid of toxicity, then it can be dangerous. Phytochemicals are concentrated pure compounds that can exhibit toxicity similar to synthetic medicines.¹⁰ Hence in terms of toxicity, they should be considered on par with synthetic medicines. Therefore, the toxicological evaluation is an important protocol of herbal drug development. Toxicological studies are conducted by bioassay and provide primary data which helps in establishing the doses which are safe for human and veterinary use.

Grewia hirsute Vahl. is a shrub which is 30-40 cm in height, belonging to family Tiliaceae. In India, 40 species of the genus were identified among which few have medicinal value. Ayurvedic scholars identify Grewia hirsuta Vahl to be belonging to Nagbala species.¹¹ In this study, we have taken the leaves and subjected to phytochemical and toxicological evaluation. 

Materials and Methods

Procurement and Authentication

The research plant was collected from natural habitat of Chittoor, Andhra Pradesh, India. The taxonomic identification of the plant was confirmed by Dr. K. Madhava Chetty, Plant Taxonomist (IAAT:357) Assistant Professor, Department of Botany, Sri Venkateswara University, Tirupati, Andhra Pradesh with a voucher number 072.

Ethics Approval

The institutional animal ethical committee approved the protocol with registration no. 112/PO/Re/S/99/CPCSEA, dated on 21/02/2019

Physiochemical Studies

The crude powdered leaves were analyzed quantitatively for physicochemical parameters such as total ash value, acid insoluble ash value, water soluble ash value, and sulfated ash value. The crude drug was subjected to various tests to determine the content of ethanol soluble, water soluble and petroleum ether extractive values along with moisture content.¹²

Preparation of Extracts

The crude powdered drug was subjected to continuous hot extraction using a Soxhlet apparatus in the order of petroleum ether, chloroform, hydroethanol and aqueous. The extracts obtained were filtered, following concentration and dried. The dried extracts were stored in airtight container at cool temperature. The extracts were subjected to quantitative phytochemical investigation with instrument FTIR, UV-visible spectroscopy and HPLC determination.¹³

FT-IR Spectroscopic Analysis

Fourier Transform Infrared Spectrophotometer (FT-IR) of hydroethanolic and aqueous extract was performed using 100 mg of the extract with a pellet approach in dried potassium bromide (300 mg) and compressed into thin tablets. They were subjected to FT-IR scan in the range of 4000-400 cm^{-1. 14}

Quantitative UV-VIS Spectroscopic Analysis

Total Phenolic Content

The total phenolic content was estimated by the folinciocalteau assay. In this test, folin-ciocalteau reagent 1 ml was mixed with 1 ml of extract and five minutes after mixing, 10 ml of 7% sodium carbonate solution was added to the mixture. 13 ml of deionized water was added and mixed thoroughly. The admixture was stored for 90 minutes for 22o C in dark condition. The admixture absorbance was measured at 650 nm. The estimation of phenolic content was carried out using a standard calibration curve of catechol solution. Total phenolic content was expressed as μg in catechol content per gram of dried extract and was estimated using formula C= C₁ x V/m; Where, C=Total phenolic content in μg/g (Catechol equivalent), C₁ = Concentration of catechol established from the calibration curve in mg/ml, V= volume of extract in milliliter, and m=the weight of the plant extract in grams.¹⁵

Tannin Content

Tannin content was estimated using 0.5 ml folinciocalteau phenol reagent. 0.1 ml of extract was mixed with 7.5 ml of deionized water in a volumetric flask to make up to volume 10 ml. The admixture was well mixed and kept in dark at room temperature for 30 minutes along with reference standard solutions of tannic acid (10, 20, 30, 40, 50 µg/ml). The absorbance for standard solution was used to make a standard curve at 700 nm. The absorbance of test solution was measured using the standard curve. The tannin content was expressed as mg/g of dried extract.

Flavonoid Content

Quercetin solution of 100 µg/ml was prepared. The concentrations of 25, 50, 75, 100, 125 and 150 µg/ml were used for the study. To each test tube, 0.5 ml extract of 0.3 ml of 5% sodium nitrite was added and was kept aside for 5 min. Then 0.3 ml of 10% aluminum chloride and 2 ml of 1M sodium hydroxide were mixed and the volume was made up to 10 ml with distilled water. The solutions were mixed well and the absorbance was measured against the blank at 510 nm.^16-17

HPLC determination of Phenols, Flavonoids and Tannins

The mobile phase A 0.1% ortho phosphoric acid and mobile phase B acetonitrile were used and run on the Phenomenex C18 column (250 X 4.6 mm, 5μ ID) at a flow rate 0.8 ml/min. 1 mg of extract was dissolved in acetonitrile and diluted to 10 ml volume in metric flasks with the same solvent. Then solution was injected directly to HPLC. HPLC chromatograms were detected using a photo diode array UV detector at wavelength 275 nm according to absorption maxima of analyzing compounds. Each compound was identified by its retention time and by spiking with standards catechol, quercetin and tannic acid under similar conditions. The peaks obtained were used for quantitative estimation of phenolic compounds, flavonoids and tannins. The quantification of the sample was done with the measurement of integrated peak area and the content was calculated using the calibration curve by plotting peak area against the concentration of the respective standard sample.^18,19

Oral Acute Toxicity Studies

Acute oral toxicity studies were carried out as per OECD guideline-425. A maximum of five animals were used in the sequential test approach. This protocol has an advantage of using the least number of animals for the estimate of acute toxicity and LD50. The hydroethanolic extract of Grewia hirsuta (HEEGH) was administered at a dose 2 g/kg orally in healthy, young adult albino Wistar female nulliparous rats (150-200 g). In the first instance, the HEEGH extract of 2 g/kg was administered to a single animal and was observed for one day to learn survival. Subsequently, the HEEGH was administered to the remaining four animals. The animals were kept under observation for every five minutes for 30 minutes, and periodically for every 2 hours for the first 24 hours. The animals were observed for 14 days following administration of HEEGH extract.²⁰

Mean body weight analysis

The body weight of animals was recorded shortly before the administration of the test substance and at the end of each week. The percentage of body weight change was calculated according to the following equation:

Relative organ weight (ROW) analysis

Percentage of body weight = × 100 Body weight at the end of each week - Initial body weight Initial body weight Relative organ weight analysis was computed as increase or decrease in organ weight to the body weight and was expressed as weight gain or loss per 100 g of body weight using the equation:^21,22

Analysis of Serum Biochemistry

Row = × 100 Absolute Organ Weight (g) Body Weight of Rats on Sacrifice day (g) Biochemistry was estimated for serum glucose, total cholesterol, triglycerides, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total protein, and blood urea nitrogen.^23,24

Hematological Analysis

Hematological analysis was carried out for hemoglobin, red blood cells, white blood cells and differential count using an automated hematology analyzer (Sysmex KX21, Japan).²⁵

Histopathology Evaluation

Histopathological evaluation of vital organs like brain, liver, kidney, heart, spleen and adrenal glands was carried out on the 15th day by sacrificing the animals. The organs were removed from the animal by abdomen midline incision method and these organs were kept in 10% buffered formalin solution for histological analysis.²⁶

Results

The percentage yield of petroleum ether extract was 3.4%, chloroform extract 2.5%, hydroethanolic extract 19.03% and aqueous extract was 2.21%. The qualitative investigations indicated numerous pharmacological active ingredients like carbohydrate, phenolic compounds, flavonoids, tannins, glycosides, alkaloids, saponins. The present study confirms the presence of phenols, phenolic acids, flavonoids and tannins by FTIR, UV-VIS spectroscopy and HPLC. The quantitative analysis data are presented in table 2 and 3.

Physicochemical Evaluation

The quantitative study revealed the physicochemical evaluation values as given in table 1.

Functional group identification

The extract absorbance band analysis in bioreduction process was observed in the region between 4000-400 cm^-1. The results indicate the phenolic hydroxyl and phenolic acid groups as primary functional groups present in HEEGH which are present in major quantities as shown in table 2 and figure 1.

UV-Spectral and HPLC study

The UV-VIS spectrophotometric and HPLC investigations revealed total phenolic content, total tannin and total flavonoid content in hydroethanolic extract as 12.35 and 10.59 CAE µg/ml, 16.00 µg/ml and 17.95 µg/ml TAE and 15.50 µg/ml and 15.26 µg/ml QAE respectively (Table 3).

Acute toxicity study

Acute toxicity studies revealed that all the animals in the study survived and tolerated 2 g/kg dose and yielded similar results on further repetition of dosing. The cut off value was identified as 5 g/kg as per fixed dose method of OECD guidelines 425

Behavioral studies, Mean body weight and Relative organ weight

No adverse reactions or behavioral changes and changes in mean body weight were observed after administration of HEEGH at 2 g/kg body weight during the entire period of experimentation. The relative organ weight results showed no alteration of the organ weight compared to the control group (p > 0.05), which confirms that the extract has no effect of weight gain or loss at the dose administered (Figure 2, 3).

Data are expressed as mean ± standard error, (n = 5). Not significant (p > 0.05) compared with control

Values are presented as a mean ± standard error. Not significant (p > 0.05) compared with control.

Hematology and Biochemistry

The results of blood and liver biomarkers studied are given in figure 4 and 5. The parameters were evaluated to be not statistically significant when compared with control (p>0.05). There were no major changes observed between control and study group at 2 g/kg. However, there were some exceptions which are shown in figure 4 and 5.

Values are presented as a mean ± standard error. Not significant (p > 0.05) compared with control.

Values are presented as a mean ± standard error. Not significant (p> 0.05) compared with control.

Histopathology of Vital Organs

Histopathology of Vital Organs The gross anatomical observations in brain, heart, liver, kidney, spleen and adrenal glands revealed no statistically significant changes when compared to control group (Figure 6-10).

Statistical Analysis

Experimental results were presented as mean ± standard error and the statistical significance between the groups were analyzed by means of one-way ANOVA followed by Dunnet’s multiple comparison test.

Discussion

Medicinal plants are important resources for new drug discovery and the advancement of therapeutics. There is a need to use latest technology which gives evidence and knowledge about phytochemicals present in medicinal plants.²⁷ The proximate analysis indicated the foreign matter 0.016% w/w, loss on drying 8% w/w and total solid content 92% w/w. The analysis indicated least impurity and the moisture content was in the acceptable value range. The total solid content indicates that the leaf powder was not adulterated. Ash values are helpful to determine the quality as well as purity of a crude drug, especially when the drug is present in powdered form. When it was subjected to determine the ash value content, the total ash content was found to be 7.5% w/w, water soluble ash 5.4% w/w, acid insoluble ash 3.30% w/w and sulfated ash was 1.80% w/w. These values match with the ash values of available crude drugs. 

The phytochemicals in the HEEGH were identified by functional chemical entity. FTIR analysis showed a prominent peak at 1396.95 cm^-1 which is finger print region for phenol and alcohol containing compounds. The flavonoids and tannins contains phenolic compounds which possess antioxidant activity. The diverse therapeutic activity against chronic oxidative stressrelated diseases like cardiovascular diseases, cancer, and deaths from all causes which stems from oxidative stress should be attributed to antioxidant nature. The confirmation by FTIR, UV and HPLC are in line with the finding of presence of phenolic compounds. The safety of the extract was established by comparison of the control with extract treated group. The behavioral, body weight and relative organ weight studies did not show any deviation from the control group (p> 0.05). No significant difference (p> 0.05) was observed when the biochemical studies and hematological parameters were compared with control group. These results were confirmed by histopathological studies, the findings of which are depicted from figure 6 to 6.4. In order to establish the dose of the extract of Grewia hirsuta, toxicity studies were conducted and it was found to be non-toxic at 2 g/kg.

 Conclusion

Phytochemical investigations established by FTIR, UV and HPLC analysis confirms the presence of phenolic compounds; flavonoids and tannins were in significant quantity. These phytochemicals are known for their antioxidant properties. The results of our study suggest that the plant is a rich source of antioxidant molecules. The extract is safe and non-toxic to vital organs, and does not interfere with hematological and biochemical reactions in vivo. From our study, it was established that HEEGH does not show any toxicity at administrated dose of 2 g/kg.