INTRODUCTION:

Herbal and herbal extracts have taken an important place in modern medicine, because of their chemical and medicinal content found in natural form. Over the last few years, the Food and Drug Administration (FDA) has approved 3 herbal treatments (anti-allergic, anticancer, and anti-psoriatic); it could change how traditional medicine is perceived in the world¹. In traditional herbal therapy, different parts of Byttneria herbacea are used as an ethanomedication to cure dysentery, leprosy, and limb fractures and relieve asthma and inflammation. The root paste is used on the wound and is taken orally to alleviate bodily pain, the leaf is used for dysentery and impaction, and the root is used to control swelling^2–4.

In the Indian health care system, it is used in panic diarrhea, bronchitis, and dysentery. As there is no relevant information about the histological character of this plant, our efforts have been focused on studying phytochemical properties and some biological activities such as the antioxidant activity of this plant.

The oxidative harm due to reactive oxygen species to proteins, lipids, and nucleic acids can cause numerous persistent diseases, which include coronary heart disease, cancer, aging, and athero- sclerosis. Antioxidants are often used in oils and fats to delay their self-oxidation. Butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), two synthetic antioxidants that were once widely employed, are now toxicologically questionable. These need to get replaced with herbal antioxidants due to the fact they were proven to be toxic and carcinogenic in animal models. It is consequently crucial to discover new assets of secure and cost-effective herbal antioxidants^5–7. The high intake of plant phenolics in the day-to-day regimen protects against cardiovascular diseases and cancer. The bioactive principles of phenolic compounds have been investigated in many in vitro studies, demonstrating their ability to suppress, for example, oxidation of low-density lipoprotein, platelet aggregation, tumor cell growth, and inflammation responses^8,9. Phenolic mixtures are admirable oxygen radical scavengers as the phenolic radical has a lower reduction potential than oxygenated radicals as well, phenoxy radicals are usually less active than oxygenated radicals^10,11. Thus, phenolic compounds may retrieve reactive oxygen intermediates deprived of triggering further oxidation reactions.

Various methods to quantify the total phenolic content of the foodstuffs or biotic analytes rely on the reaction of phenol with a colorimetric reagent to allow measurements in the visible spectrum. The Folin–Ciocalteu (F-C) assay is one such process and has been projected as a standardized technique for utilization in repetitive quality control and assessment of the antioxidant potency of food and nutritional supplements. The F-C test relies on the removal of electrons from phenolics into phosphotungstic/phosphomolybdic complexes in an alkaline medium to form a blue complex, predictable by spectroscopy at around 760 nm^12–14.

MATERIAL AND METHODS:

Plant Collection and Authentication:

Byttneria herbacea plants were collected from the Sambalpur of the Odisha region. Herbariums were produced and authenticated in the Botanical Survey of India, Central National Herbarium, Howrah-711103. The herbarium was kept in the museum and the specimen number is given: SKK-01.

Crude Drug Extraction:

Aerial parts of Byttneria herbacea were dried in shade and powdered in a domestic mixer. The coarsely powdered drug was extracted with Petroleum Ether in the continuous hot percolator. The insoluble residue was dried and further extracted with ethanol. Extracts were prepared using Petroleum Ether (60⁰-80⁰C) and Ethanol. The extracts were concentrated in the Rotary evaporator under vacuum conditions and kept in the refrigerator at temp. 2⁰C to 4⁰C.

Phytochemical Screening:

A Judicious quantity of extracts was used for phytochemical screening for the compound as total phenol, alkaloids, flavonoids, tannins, saponins, glycosides, etc. As described in standard reference books with little modification, 2.0gm of plant extract was diluted in 10ml double-distilled water and filtered with Whatman filter paper, and further utilized for phytochemical screening^7,15–18.

Test for Alkaloids:

Wagner's Test: Wagner's reagent was added to 1ml of the extract. The formation of a reddish-brown precipitate indicates the presence of alkaloids.

Dragendorffs Test: 1ml Dragendorffs reagent was applied to 1 ml extract, and an orange-red precipitate indicated the existence of alkaloids.

Test for Tannins:

A few drops of ferric chloride solution (5%) were added to approximately 1ml of extract. The emergence of a greenish-black precipitate suggested the presence of tannins.

The presence of a creamy precipitate with a 10% lead acetate reagent indicated a positive tannin test.

Test for Glycosides:

Keller-Kiliani Test: After thoroughly mixing the dried extract in glacial acetic acid, a few drops of ferric chloride solution and a few drops of concentrated sulfuric acid were added. The formation of a greenish-blue precipitate indicates the presence of glycosides.

Legal’s Test: The dried extract was treated with 1 ml of pyridine and a few drops of sodium nitroprusside solution. Sodium hydroxide solution was added to make it alkaline. Pink to crimson coloration indicates the presence of glycosides.

Test for Flavonoids:

Shinoda’s Test:

A few fractions of the extract were mixed with 5ml of 95% ethanol and a few drops of concentrated hydrochloric acid. Following the addition of the magnesium turnings (0.5g), a drop-wise addition of concentrated hydrochloric acid was made followed by the heating. The presence of flavonoids is indicated by the presence of magenta color.

Test for Saponins:

A dilute aqueous solution of the extracts was agitated for 15 minutes in a test tube. The presence of saponins is indicated by the development of foam.

Test for Phenols:

A small fraction of the extract was dissolved in about 1ml of distilled water in a test tube. A bluish-green color was developed after the addition of 2 drops of Ferric chloride solution indicating the presence of Phenols.

Test for Terpenes:

Liebermann Burchards Test:

The extract was treated with a few drops of acetic anhydride and 1ml of concentrated sulphuric acid was added from the sides of the test tube. A violet ring was formed at the junction of two layers indicating the presence of terpenes.

Test for Triterpenes:

Chloroform extract of the extract was treated with concentrated sulphuric acid (H₂SO₄). The appearance of a reddish-brown ring indicated the presence of triterpenes¹⁹.

Sedimentation Test:

To the extract, a few drops of 10% Lead acetate solution were added. The formation of a precipitate indicates the presence of triterpenes²⁰.

Color Test:

To the extract, one drop of 10% Copper sulfate solution and 1 ml of Sulphuric acid were added. The appearance of the blue-green color on heating indicates the presence of triterpenes²⁰.

Total Phenolic Content:

The total phenol contents of ethanolic extracts (1mg/1ml) of Byttneria herbacea were determined spectrophotometrically by diluting 100µl of the sample solution with 400µl distilled water, adding 150µl of Folin-Ciocalteu reagent(ACS Chemicals, Ahmedabad) (1:1 dilution), and 500µl of 20% Na₂CO₃ solution after 5 minutes. After thoroughly mixing the contents, they were left in the dark for one hour, and then at 765 nm, the absorbance was measured by using UV-Spectrophotometer (UV-2600i, Shimadzu). Simultaneously a standard graph was obtained by using 1 ml aliquots of Gallic acid (Marc Flavours, Greater Noida) as standard (50-450µg/ml) and by taking the absorbance at 765 nm¹³.

For the determination of total phenolics, the equation C=C₁×V/m was used. C = the total phenol content as GAE in mg/g, C₁ = the Gallic acid concentration recognized from the standard graph in mg/ml, V = volume in ml, and m = the total weight of extract in g, respectively^21–24.

Antioxidant activity:

The antioxidant activity of the extracts was determined by the method based on the hydrogen donating capacity or scavenging activity of extracts on the DPPH (Sisco Research Laboratories Pvt. Ltd, Andheri, Mumbai) free radicals^10,25,26. The methanolic solutions of the extracts of Byttneria herbacea (3ml) at different concentrations were mixed with 4ml of a 0.004% methanolic solution of DPPH. Methanolic solution of DPPH (0.0004%) was used as control and methanol as blank. Absorbance at 517nm was measured by using UV-Spectrophotometer (UV-2600i, Shimadzu) after 30 minutes of reaction^27,28.

The absorbance of the methanolic solution of the extract was plotted against corresponding concentrations and compared with the standard anti-oxidant Gallic acid^29–31. The percentage DPPH decolorization by the sample was calculated as:

% Decolourization = (1-A/A₀) x 100

Where A₀is the absorbance of DPPH without the sample (control) and A is the absorbance of the sample containing DPPH at 517nm.

The % decolorizations against the extract concentrations were plotted, to obtain the logarithmic regression line for estimation of the IC₅₀(Inhibitory concentration 50, µg/ml), the quantity of extract required to drop the absorbance of DPPH by 50%. The IC₅₀ was calculated by using the parabolic equation y= ax²+ bx + c, for the value of y=50

RESULTS:

Phytochemical Screening:

The experiments conducted in Byttneria herbacea for phytochemical constituent analysis showed the results summarized in table 1.

Table 1: Phytochemical analysis of the ethanol extract of the areal parts of Byttneria herbacea

Test for	Reagent	Ethanol Extract
Alkaloids	Wagners Reagent	+
Alkaloids	Dragandroff’s Reagent	+
Tannins	Ferric Chloride	+
Tannins	Lead acetate	+
Glycosides	Keller-Kiliani Test	+
Glycosides	Legal’s Test	+
Flavonoids	Shinoda Test	+
Saponin	Water	+
Phenol	Ferric Chloride	+
Terpenes	Liebermann Burchards Test	+
Triterpenes	H₂SO₄ Test	+
	Sedimentation Test	+
	Color Test	+

(+) shows presence

Total phenolic content:

Phytochemical screening of the ethanolic extracts of Byttneria herbacea was performed, and the extracts showed the presence of phenolic compounds. Using the F-C technique, the total phenolic content was estimated as the Gallic acid equivalent (GAE) expressed in mg/g. By using the graph in fig. 1, the total phenolic content of the extract was calculated. The equation of the standard graph was y = 0.0012x - 0.0413, with R² = 0.9929. It was revealed that the extracts had a total phenolic content of 185.14 mg/g (expressed as Gallic acid equivalents, GAE).

Antioxidant activity:

The phenol content is directly interrelated to the antioxidant activity. In fig. 2, the DPPH activities of the sample are shown. The % decolorization of different concentrations of the sample was plotted and the equation of the graph was found to be y = -0.0004x² + 0.2924x + 5.0154. The IC₅₀was calculated from the value of y =50 and was found to be 220.14(µmol/g).

Figure 1: Standard curve of Gallic acid

Figure 2: Activity (Decolorization %)

DISCUSSION:

In this study, we chose the plant Byttneria herbacea of the Malvaceae family to investigate its phytoconstituents and biological activity, such as antioxidant activity, based on its ethnomedicinal use by tribal peoples in various regions of India. The plant was discovered to be a rich source of phenols and to have significant antioxidant activity. The Folin-Ciocalteu reagent was used to estimate total phenolics, and the DPPH assay method was used to determine antioxidant activity. Despite the efforts made during the work, it is believed that there is still room for further phytochemical studies on the plant extract using modern analytical techniques. The research can be expanded to isolate and characterize the phytoconstituents found in the extract.

Conclusion:

According to the findings of this study, the ethanol extract of the areal parts of Byttneria herbacea contains a significant amount of phenolics. The presence of an extractable phenolic compound confirms the results of antioxidant activity. As a result, it can be used as a natural source antioxidant with great potential as a therapeutic agent in preventing or slowing the progression of aging and age-related oxidative stress-related degenerative diseases.

REFERENCES:

1. Aarland RC. Peralta-Gómez S. Sanchéz CM. et al. A pharmacological and phytochemical study of medicinal plants used in Mexican folk medicine. Indian J Tradit Knowl. 2015;14(4):550–557. Toegang verkry Junie 29, 2021. www.medicinaltradicionalmexicana.unam.mx

2. Somkuwar SR. Chaudhary RR. Chaturvedi A. Preliminary phytochemical screening of Byttneria herbacea Roxb . 2014;1(2).

3. Whitlock BA. Hale AM. The phylogeny of Ayenia, Byttneria, and Rayleya (Malvaceae s. l.) and its implications for the evolution of growth forms. Syst Bot. 2011;36(1):129–136. doi:10.1600/036364411X553216

4. Somkuwar SR. Taxonomic Update and Habitat Status to Byttneria herbacea from Peninsular India. Adv Zool Bot. 2020;8(4):326–333. doi:10.13189/azb.2020.080404

5. Odabasoglu F. Aslan A. Cakir A. et al. Comparison of antioxidant activity and phenolic content of three lichen species. Phyther Res. 2004;18(11):938–941. doi:10.1002/ptr.1488

6. Li HB. Cheng KW. Wong CC. Fan KW. Chen F. Jiang Y. Evaluation of antioxidant capacity and total phenolic content of different fractions of selected microalgae. Food Chem. 2007;102(3):771–776. doi:10.1016/j.foodchem.2006.06.022

7. Valli G. Jeyalakshmi M. Preliminary Phytochemical and Antioxidant Study of Odina woodier Leaf Extract. 2012;2(4):153–155.

8. Kokko HI. Cultivated sweet rowanberries have high phenolic content and antioxidant capacity Antioxidant Capacity and Phenolic Content of Sweet Rowanberries. 2007;(January).

9. Tiwari P. Patel RK. Total phenolics and flavonoids and antioxidant potential of Draksharishta prepared by traditional and modern methods. Pharmacologyonline. 2011;3(1):1072–1082.

10. Krishna KV. Karuppuraj V. Perumal K. Antioxidant activity and Folic acid content in indigenous isolates of Ganoderma lucidum . Asian J Pharm Anal. 2016;6(4):213. doi:10.5958/2231-5675.2016.00032.6

11. Senthil Kumar M. Balachandran S. Chaudhury S. Influence of Incubation Temperatures on Total Phenolic, Flavonoids Content and Free Radical Scavenging Activity of Callus from Heliotropium indicum L. Pharm Res. 2012;2(4):148–152. www.asianpharmaonline.org

12. Ainsworth EA. Gillespie KM. Estimation of total phenolic content and other oxidation substrates in plant tissues using Folin-Ciocalteu reagent. Nat Protoc. 2007;2(4):875–877. doi:10.1038/nprot.2007.102

13. Tiwari P. Patel RK. Estimation of Total Phenolics and Flavonoids and Antioxidnt Potential of Ashwagandharishta Prepared by Traditional and Modern Methods. Asian J Pharm Res. 2013;3(4):1147–1152.

14. Samydurai P. Saradha M. Effects of Various Solvent on the Extraction of Antimicrobial, Antioxidant Phenolics from the Stem Bark of Decalepis hamiltonii Wight and Arn. . Asian J Res Pharm Sci. 2016;6(2):129. doi:10.5958/2231-5659.2016.00018.7

15. WHO. Quality control methods for medicinal plant materials World Health Organization Geneva. Who. Published online 1998. doi:10.1016/j.pbi.2015.01.003

16. Wadood A. Phytochemical Analysis of Medicinal Plants Occurring in Local Area of Mardan. Biochem Anal Biochem. 2013;02(04):Ferrannini, Ele, and Ralph A. DeFronzo. 2015. “Imp. doi:10.4172/2161-1009.1000144

17. Vaghasiya Y. Dave R. Chanda S. Phytochemical analysis of some medicinal plants from western region of India. Res J Med Plant. 2011;5(5):567–576. doi:10.3923/rjmp.2011.567.576

18. Ali S. El-Ahmady S. Ayoub N. Singab A. Phytochemicals of Markhamia Species (Bignoniaceae) and Their Therapeutic Value: A Review. European J Med Plants. 2015;6(3):124–142. doi:10.9734/ejmp/2015/15015

19. Harborne JB. (1998) Textbook of Phytochemical Methods. A Guide to Modern Techniques of Plant Analysis. 1998;3rd Editio:4–7.

20. Shanaida M. Pryshlyak A. Golembiovska O. Determination of triterpenoids in some lamiaceae species. Res J Pharm Technol. 2018;11(7):3113–3118. doi:10.5958/0974-360X.2018.00571.1

21. Sanjeeb KK. Shubhrata N. NainaP. Asutosh M. Quantification of Phenol Content and Antioxidant Activity From Ziziphus Jujuba (Family: Rhamnaceae). J Harmon Res ( JOHR ). 2013;2(4):222–225.

22. Aher N. Chaudhari S. Zalte A. Morphological and microscopical studies and phytochemical analysis of Markhamia falcata (Seem). Res J Pharm Technol. 2020;13(3):1117–1120. doi:10.5958/0974-360X.2020.00205.X

23. Parejo I. Viladomat F. Bastida J. et al. Investigation of Bolivian plant extracts for their radical scavenging activity and antioxidant activity. Life Sci. 2003;73(13):1667–1681. doi:10.1016/S0024-3205(03)00488-0

24. Siddiqui N. Rauf A. Latif A. Mahmood Z. Spectrophotometric determination of the total phenolic content, spectral and fluorescence study of the herbal Unani drug Gul-e-Zoofa (Nepeta bracteata Benth). J Taibah Univ Med Sci. 2017;12(4):360–363. doi:10.1016/j.jtumed.2016.11.006

25. Talluri MR. Gummadi VP. Battu GR. Antioxidant Activity of Borassus Flabellifer. Int Res J Pharm. 2017;8(3):18–22. doi:10.7897/2230-8407.080329

26. Kedare SB. Singh RP. Genesis and development of DPPH method of antioxidant assay. J Food Sci Technol. 2011;48(4):412–422. doi:10.1007/s13197-011-0251-1

27. Raj PS. Preeth M. Shobana J. Minaz N. Nazeer A. In Vitro Anti-Oxidant Activity of the Various Extracts of Tribules terristeris Leaves by UV Spectrophotometer. 2010;1(2):152–155.

28. R M. Antioxidant activity of extract from the leaves of Tylophora asthmatica. J Microbiol Antimicrob. 2012;4(4):80–82. doi:10.5897/jma11.104

29. Madhu C. Swapna J. Neelima K. Shah M V. Linn A. RESEARCH ARTICLE A Comparative Evaluation of the Antioxidant Activity of Some Medicinal Plants Popularly Used in India. 2012;2(3):98–100.

30. Jadhav GB. Saudagar RB. Free radical Scavenging and Antioxidant Activity of Punica granatum Linn. Asian J Res Pharm Sci. 2014;4(2):5.

31. Li X. Wu X. Huang L. Correlation between antioxidant activities and phenolic contents of Radix Angelicae Sinensis (Danggui). Molecules. 2009;14(12):5349–5361. doi:10.3390/molecules14125349

Received on 04.12.2021 Modified on 10.04.2022

Research J. Pharm. and Tech 2023; 16(4):1659-1663.

DOI: 10.52711/0974-360X.2023.00271