P. Vishnu Mohan Reddy¹, Pulala Raghuveer Yadav², A. Lakshmi Devi³, Lepakshi Md. Bhakshu⁴ and K. Venkata Ratnam^1*

¹Department of Botany, Rayalaseema University, Kurnool, 518 007, Andhra Pradesh, India.

²Department of Biotechnology, Indian Institute of Technology Hyderabad, Kandi 502 285, Telangana State, India.

³Department of Botany, S.V.B. Government Degree College, 518 134, Andhra Pradesh, India.

⁴Department of Botany, PVKN Government College (A), Chittoor, 517 002, Andhra Pradesh, India.

Corresponding Author E-mail: drvenkatapkd@gmail.com

DOI : https://dx.doi.org/10.13005/bpj/2870

Abstract

The objective of the present investigation is to appraise the qualitative and quantitative phytochemical analysis, DPPH and Hydroxyl radical scavenging activity along with a total antioxidant capacity of water extract obtained from six medicinal plants, i.e., Acalypha alnifolia,  Caesalpinia bonduc, Carissa spinarum, Commiphora caudata, Moringa concanensis and Terminalia tomentosa. The phytochemical analysis of the six medicinal plants water extracts revealed total phenolic content (TPC) in the range of 27.77 to 3.35 GAE mg/g dwt. The highest TPC is present in A. alnifolia and the lowest is noticed in T. tomentosa. The total flavonoid content is present in between 1.68 to 1.48 QE mg/g dry weight and a higher amount of flavonoid content was observed in A. alnifolia and lowest present in C. bonduc. Antioxidant activity results of the six medicinal plants showed that the highest total antioxidant capacity was observed in Carissa spinarum and lowest in T. tomentosa. DPPH method was used to know the antioxidant capacity of aqueous extract of the six medicinal plants. Among the tested plants A. alnifolia, Carissa spinarum and Moringa concanensis strongly reduced DPPH purple color by expressing ~80% as maximum inhibition. DPPH was strongly inhibited by T. tomentosa (IC50 value 25 μg/ml). Hydroxyl radical was strongly (~96%) inhibited by T. tomentosa and lowest IC50 value was expressed by A. alnifolia (36.4 μg/ml). The study results indicate that all six medicinal plants are rich sources of natural antioxidant components.

Keywords

Aqueous Extract; Antioxidant Studies; Eastern Ghats

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Copy the following to cite this article: Reddy P. V. M, Yadav P. R, Devi A. L, Bhakshu L. M, Ratnam K. V. Phytochemical Analysis of Selected Medicinal Pslants from Eastern Ghats of Andhra Pradesh. Biomed Pharmacol J 2024;17(1).

Copy the following to cite this URL: Reddy P. V. M, Yadav P. R, Devi A. L, Bhakshu L. M, Ratnam K. V. Phytochemical Analysis of Selected Medicinal Pslants from Eastern Ghats of Andhra Pradesh. Biomed Pharmacol J 2024;17(1). Available from: https://bit.ly/3wL9p7y

Introduction

Free radicals are fragments of molecules with a very short half-life, generated through internal/ external sources, are highly reactive and damage macromolecules like proteins, DNA and lipids of cell membranes of living organisms. The most common reactive oxygen species are hydroxyl (OH), hydrogen peroxide (H2O2), super oxide anion (O2), and peroxyl radicals (ROO) and nitrogen derived free radicals are nitric oxide (NO), peroxyl nitrite anion (ONOO), Nitrogen dioxide (NO2) and Di-nitrogen trioxide (N2O3). The chemical constituents that retard or suppress oxidation or prolong the life of the oxidizable molecules/ inhibit the oxidation process are called antioxidants. Antioxidants from natural/plant sources enhance the endogenous enzymes’ anti-oxidative capability and reduce the risk of many free radical mediated diseases. Traditional medicinal systems use medicinal plants containing various chemical components such as alkaloids, polyphenols, glycosides and terpenoids, which showed pharmacological properties such as antioxidant and antimicrobial activities ^{1, 2}. Of the estimated 2.5 to 5 lakhs plant species; very few have been screened for its biological or pharmacological activities³. In this connection, we selected the following water extract of six medicinal plants to screen phytochemical analysis and antioxidant potential. 

Acalypha alnifolia Klein ex Willd., is a rare medicinal plant found in the forests of South India⁴. Acalypha belongs to the family Euphorbiaceae and is the fourth largest genus with about 450 species^5,6. In traditional medicine, the plant has been used to treat dysentery, diabetes and as mosquito repellent ^4,7,8. 

Caesalpinia bonduc Roxb. (family: Caesalpiniaceae) is locally known as Lata Karanja, is a prickly woody liana distributed throughout the hotter parts of India and Sri Lanka ⁹ It is a valuable medicinal plant and its different parts, such as bark, leaves, roots and seeds are utilized in traditional system of medicine. The roots are effective as an antiperiodic and antispasmodic properties¹⁰, the bark is a good remedy as anthelmintic and febrifuge and the leaves were reported as an emmenagogue¹¹. The seeds are reported to have various pharmacological actions like antipyretic, antiperiodic, asthmatic and febrifuge ^{12, 13}. 

Carissa spinarum L. is a spinous evergreen shrub that belongs to the family Apocynaceae, distributed throughout dry localities in India. The plant has been used in the Ayurvedic medical system to treat liver problems, epileptic disease, microbial infections, cytotoxic, viral diseases, inflammation, arthritis and cancer¹⁴. The ripened fruits are edible and reported to have cardiac protective properties. 

Commiphora caudata (Arn) Engl. belongs to the family Burseraceae, is a thorny shrub to medium sized tree distributed in dry deciduous forests of Peninsular India of Andhra Pradesh, Karnataka and Tamil Nadu ^{15, 16}. It is commonly known as a hill mango because of the mango smell of its stem bark. Traditionally, the bark has been used to treat diabetes, arthritis and obesity ^{17, 18}. Fruits have been used to prepare pickles and gum is used as incense.

Moringa concanensis Nimmo, is a rare medicinal plant of the family Moringaceae. It is found in dry localities in Konkan, Andhra Pradesh. Telugu is called Konda munaga/ Karumunaga, because of its similar morphological characters of drumstick plant M. oleifera¹⁹. Different parts of this plant have been reported to treat different human ailments such as leaves for gynic problems, hyper-tention, constipation, jaundice, skin cancers, diabetes and splenomegaly, stem bark for abortion and fruits for rheumatism, nervous disorders, curing liver and spleen diseases, gum for dental problems and flowers for aphrodisiac, leucorrhea and abortion²⁰. 

Terminalia tomentosa is a deciduous tree that belongs to the family Combretaceae. Generally, T. tomentosa is called a “crocodile bark tree” because the bark of this has characteristics feature like the skin of crocodile. It is found in Southern parts of the Indian subcontinent and other Southeast Asian countries²¹. In Ayurveda, the bark treats rheumatism, fever, urinary diseases and diabetes, vertigo, piles, constipation and chronic dysentery²².

Materials and methods

Plant materials

Selected medicinal plant parts i.e. A. alnifolia (aerial parts), C. bonduc (seeds), C. spinarum (fruits), C. caudata (stem bark), M. concanensis (leaves) and T. toemntosa (stem bark) were collected from Nallamala forests of Kurnool District, Andhra Pradesh, India. (Fig.1). The voucher specimens were deposited in Rayalaseema University Herbarium, Kurnool, Andhra Pradesh, India and were identified with the help of regional ²³ and local floras ²⁴.

Figure 1: Photographs of the selected medicinal plants Click here to view Figure

Preparation of water extracts

The plant parts were cleaned with tap water, sliced and shade dried. The mixer grinder was used to grind the dried material to fine powder. The powdered material was mixed with double distilled and boiled for 30 min and filtered. The filtrate was concentrated on water both. The phytochemical and antioxidant activities of crude water extract (WE) were analyzed.

Preliminary Phytochemical Screening

Qualitative phytochemical screening of the selected six medicinal plant extracts was analyzed using standard methods ²⁵.

Total phenolic content

Folin-Ciocalteu (FCA) reagent method was used to estimate the total phenolic content of the water extracts in the six medicinal plants²⁶. The detailed procedure was followed as described ²⁷.

Total flavonoid content

Aluminum chloride method was used to estimate the total flavonoid content (TFC) of the water extracts of the six medicinal plants²⁸. The detailed procedure was followed as described ²⁷.

Ammonium molybdate dependent antioxidant capacity

Ammonium molybdate dependent total antioxidant capacity of the selected medicinal plant extracts was estimated by²⁹. The detailed procedure was followed as described²⁷.

DPPH and Hydroxyl radical scavenging activity

DPPH and Hydroxyl radical activity of water extracts were measured as mentioned in detail elsewhere ^{30, 31}. The detailed procedure was followed as described ²⁷.

Results

Qualitative Phytochemical Screening

The preliminary phytochemical studies on six selected medicinal plants revealed the presence of ten different secondary metabolites. Among the tested components, alkaloids, flavonoids, terpenoids, phenolic, tannins and glycosides showed strong reactions.  At the same time, steroids showed very feeble reactions. Anthraquinones showed negative results in the tested plants.

Quantitative estimation of total phenolic/flavonoid content

The results on total phenolic content in the selected medicinal plants are between 190 to 25 GAE mg/g dwt. The highest TPC is present in TT and the lowest is noticed in CB (Figure 1). The total flavonoid content is between 80 to 12 QE mg/g dwt. The flavonoid content was higher in TT and lowest present in CB (Figure 2). Gallic acid and quercetin were used as standards.

Figure 2: Total Phenolic (A) and Flavonoid content (B) of water extract of selected medicinal plants Click here to view Figure

Total antioxidant capacity (TAC)

The results on TAC of the selected medicinal plant extracts indicate that they expressed 240 to 40 ASE mg/g dwt. The highest TAC was observed in TT and the lowest in CB (Figure 3). Ascorbic acid was used as a standard component.

Figure 3: Total antioxidant capacity (TAC) of water extract of selected medicinal plants Click here to view Figure

DPPH scavenging activity

The results revealed that all the plants expressed concentration dependent DPPH quenching activity. The plants, AA, CS and MC reduced DPPH purple color strongly by expressing ~80% as maximum inhibition. The extracts exhibited IC₅₀ values to inhibit 50% of the DPPH radical between 300 to 25 μg/ml (Table 1).

Figure 4: DPPH quenching activity of water extract of selected medicinal plants Click here to view Figure

Hydroxyl radical scavenging activity

All the plants tested exhibited concentration dependent hydroxyl radical scavenging activity (Fig. 5). Among the test plants A. alinifolia showed lowest IC₅₀ value (36.4 ug/ml) and T. tomentosa showed ~96% as maximum inhibition (Table 1).

Figure 5: Hydroxyl radical scavenging activity of water extract of selected medicinal plants Click here to view Figure

Table 1: Qualitative phytochemical analysis of water extracts of selected medicinal plants.

Type of the Component	Selected Medicinal Plants
	AA	CB	CS	CC	MC	TT
Alkaloids	+++	+	++	+	+	+
Anthraquinones		–	–
Coumarins	++	+	+	+	++	+
Catecholic compounds	T	+	+	–		++
Glycosides	+	T	+	–	+	+++
Flavonoids	+	++	++	+	+++	++
Saponins	++	+	+	–	–	++
Steroids	–	–	–	T	–	–
Tannins		+	+	+	–	++
Terpenoids	+	+	–	++	+	+
Phenolic compounds	++	++	++	++	+	+++

 

Discussion

Recently researchers have been interested in exploring natural antioxidant principles that are therapeutically potent and with minimum low or no side effects to treat various human ailments and for the food industry. Plants synthesize a wide variety of chemical components such as alkaloids (nitrogenous compounds), terpenes (lipid derivatives) and phenolics (carbohydrate derivatives) with potential pharmacological properties³². Thousands of biologically active phytoconstituents are isolated from higher plants. Of which phenolic compounds are phenolic acids, vitamin E, coumarins, flavonoids, isocoumarins, biflavonols, stilbene, phenols, quinones, betacitie, and chromones etc. reported exhibiting intense antioxidant activity. Scientifically phenolic and flavonoids are reported to have various pharmacological activities such as antioxidant, antiulcer, antispasmodic, cytotoxic anti-inflammatory, antitumor, and antidepressant activities^{33 to 37}.

The Quantity of total phenolic and flavonoids of the water extracts of the selected medicinal plants is estimated using the colorimetric method. The results indicated that T. tomentosa stem bark has more phenolic and flavonoid contents. The Genus Terminalia belongs to the Combretaceae and comprises about 250 species. Of these, only 39 species were studied for its phytochemical composition and 368 phyto-constituents such as terpenoids, flavonoids, tannins, simple phenolics, phenylpropanoids, etc. Quantitative estimation of phenolic and flavonoid content from T. tomentosa stem bark was reported³⁸. They reported higher values of TPC and less TFC than the contents of the present study. Budholiya and Sharma³⁹ estimated total phenolic and flavonoid contents from T. toemntosa leaf extracts.

Vasthi Kennedy and Devarajan Natarajan ⁴⁰ studied antioxidant and phytochemical analysis of A. alnifolia leaf, especially methanol and aqueous extracts. In the present study, water extract has higher phenolic and flavonoid content than the previous report (Fig. 2A & B). TPC and TFC of C. bonduc seeds showed a lower range than previously reported results ⁴¹. This may be due to the variation in the climatic condition in both atmospheric and edaphic conditions. 

Phytochemical reports of the genus Commiphora, resulted in the identification of more than 300 chemical constituents ⁴². Very few and sporadic attempts were made on phenolic and flavonoid contents of C. caudata leaf ethanol extract ⁴³. M. concanensis a rare wild medicinal plant of tropical deciduous forest, received good attention in phytochemical reports of different parts. The results indicated the presence of volatile oils, flavonoids, alkaloids, tannins, and fatty acids ⁴⁴. Few reports were noticed on quantitative analysis of phenolics and flavonoids from M. concanensis⁴⁵. A critical review of quantitative estimation of the phytochemical composition of aqueous extracts of C. caudata (stem bark), C. spinarum (fruits), and M. concanensis (leaves) revealed that no previous report was noticed on total phenolic and flavonoid contents, hence, present report on the said species gains importance.

The standard and common method used to estimate the antioxidant capacity of medicinal plant extract is the phosphomolybdenum method. Antioxidant components reduce molybdenum and form a green coloured MO V complex. Revathi et al.,⁸ reported phosphomolybdenum dependent antioxidant capacity of A. alnifolia leaf extracts i.e., Petroleum ether (38.7 ± 2.2), methanol (139.7 ± 2.8) and hot water extract (82.9 ± 6.4 mg AAE/g). In our current study the total antioxidant capacity of water extract of A. alnifolia aerial parts showed similar values (80.13 ± 1.05 mg AAE/g) to the previously reported values. Based on the review of the antioxidant potential of aqueous extracts on ammonium molybdate dependent antioxidant capacity of C. caudata (stem bark), C. spinarum (fruits), M. concanensis (leaves) and T. tomentosa (stem bark) revealed that no previous report was noticed on ammonium molybdate dependent antioxidant capacity. Hence, the present report on the said species gains importance.

DPPH is a synthetic free radical commonly used to evaluate the antioxidant potential of herbal drugs/pure constituents. The results on DPPH reducing activity revealed that, among the test plant extracts, A. alnifolia strongly (88%) reduced DPPH purple color than other plant extracts. T. tomentosa exhibited the lowest IC₅₀ value (25 μg/ml), indicating its potential as an antioxidant. The intense DPPH quenching activity of T. tomentosa may be responsible for the presence of ellagic acid and gallic acid, which were reported as potential antioxidants/ therapeutics from different traditional medicinal plant sources⁴⁶.  Evangelene and Natarajan⁴⁷ observed the antioxidant activity of A. alnifolia leaf methanol and water extracts by the DPPH method. Here, we studied the DPPH scavenging activity of water extract of A. alnifolia aerial parts, the results demonstrate that water exhibited IC₅₀ values (42.5µg/ml) higher than leaf extracts. DPPH reducing activity of C. caudata stem bark extracts ethyl acetate, methanol, petroleum ether and chloroform was reported⁴⁸. Here we reported DPPH quenching capacity of water extract. Vijay Kumar et al., ⁴⁹ reported DPPH scavenging activity of methanol extract of M. concanensis leaf. The results indicated that ethanol and methanol extracts showed maximum inhibition of 69% and 66 % at 250 µg/ml concentration. In the present study, also reported the DPPH quenching capacity of water extract. It required 60 µg/ ml to reduce 50% DPPH purple color. A critical review of the literature indicated that no previous report was noticed on the DPPH scavenging activity of water extract of C. caudata (stem bark), C. spinarum (fruits), P. toemntosa (leaves) and T. toemntosa (stem bark).

Hydroxyl radicals formed through Fenton’s reaction are the most reactive oxidative molecules capable of damaging biological molecules/ membranes in living cells. In the present experiment, T. tomentosa exhibited maximum hydroxyl radical inhibition as ~96%. A. alnifolia showed the lowest IC₅₀ value (36.4 μg/ml), indicating its potentiality as hydroxyl radical inhibitor. Free radical scavenging property of C. caudata stem bark was reported by DPPH, nitric oxide, SOD methods by in vitro^{43, 48, 50} and in-vivo studies ⁵¹. Antioxidant activity of leaf and fruit oils of C. caudata was reported by Reddy et al.,⁵². No previous report was noticed on the hydroxyl radical scavenging capacity of C. caudata stem bark extracts by in vitro studies.

The Hydroxyl radical quenching activity of M. concanensis leaf ethanol extract was studied by Balakrishnan & Krishnasamy⁵³. The results showed that, ethanol extract expressed very feeble activity i.e. it expressed a very high IC₅₀ value (400 µg/ ml) and 70% as maximum inhibition at 500 µg/ ml concentration. In the present study, water extract exhibited significant hydroxyl radical scavenging activity by expressing a low IC₅₀ value i.e., 60 µg/ ml. Critical review on hydroxyl radical scavenging activity of A. alnifolia aerial parts⁵⁴, C. spinarum (fruits), T. tomentosa stem bark^{55, 56}, and P. tomentosa leaves ⁵⁷ was not reported by earlier workers. Hence, the present work provides additional information on the hydroxyl radical scavenging potential of above mentioned plants.           

Conclusion

The current in vitro studies indicated that water extracts of the selected medicinal plants have a high amount of phenolic and flavonoid components, good antioxidant capacity and strongly inhibited Hydroxyl radicals and reduced DPPH, The findings of the study suggest that the selected medicinal plants could be used as a potential source of natural antioxidants.

Acknowledgements

None

Conflict of Interest

Authors declared as no conflict of interest

Funding Sources

Nil

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