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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 6  |  Issue : 2  |  Page : 79-88

Botanical standardization, phytochemical analysis, and antioxidant studies of various fractions of Atibala [Abutilon indicum (L.) Sweet] leaves


1 Department of Pharmacognosy, Central Ayurveda Research Institute, CCRAS, Ministry of AYUSH, Kolkata, West Bengal, India
2 Department of Pharmacognosy, G. Pulla Reddy College of Pharmacy, Mehdipatnam, Hyderabad, Telangana, India
3 Department of Ayurveda, Regional Ayurveda Research Institute, Thiruvananthapuram, Kerala, India
4 Central Council for Research in Ayurvedic Sciences, New Delhi, India

Date of Submission21-Jul-2021
Date of Acceptance01-Dec-2021
Date of Web Publication19-Jan-2022

Correspondence Address:
Dr. Rajesh Bolleddu
Department of Pharmacognosy, Central Ayurveda Research Institute, CCRAS, Government of India, Kolkata 700091.
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jdras.jdras_8_21

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  Abstract 

BACKGROUND: Abutilon indicum (L.) Sweet (family: Malvaceae) is a perennial herb with golden yellow flowers called as Atibala in Ayurveda. The roots of this plant were widely used in traditional system of medicine as aphrodisiac, uterine tonic, and leaves are used in bronchitis, gonorrhea, fever, and urethritis. AIM: In this study, histological, powder microscopical studies of A. indicum (Malvaceae) leaves, followed by total phenolic, flavonoid content, antioxidant potential of hydroalcoholic extract, and its fractions, were determined. MATERIALS AND METHODS: Anatomical, powder microscopical studies were carried out according to the Ayurvedic Pharmacopoeia of India. Hydroalcoholic extract was subjected to fractionation with different solvents, performed phytochemical studies for all fractions, and screened for DPPH (diphenyl picrylhydrazyl) radical scavenging activity and reducing power capacity. RESULTS: The phytochemical screening revealed the presence of phenolic and flavonoid compounds in all the fractions. The total phenolic content of hydroalcoholic/mother extract and all fractions was ranged from 20 to 40 mg GAE/g. The flavonoid content of mother extract and all fractions was measured; values ranged from 16 to 30 mg RE/g. The highest DPPH radical scavenging activity was demonstrated by ethyl acetate fraction (IC50-60 µg/mL), followed by butanol fraction (IC50-95 µg/mL). The reducing powers of all the extracts were comparable with those of positive control butylated hydroxyl toluene (BHT). CONCLUSION: The high content of phenolic compounds indicated that these compounds are responsible for antioxidant activity. Therefore, ethyl acetate fraction of A. indicum leaves can be considered as a promising candidate for natural plant sources of antioxidants.

Keywords: In-vitro antioxidant activity, phytochemical analysis, powder microscopy


How to cite this article:
Bolleddu R, Venkatesh S, Mangal AK, Varanasi S, Paria D, Prasad PV, Shiddamallayya N, Bandi V, Srikanth N. Botanical standardization, phytochemical analysis, and antioxidant studies of various fractions of Atibala [Abutilon indicum (L.) Sweet] leaves. J Drug Res Ayurvedic Sci 2021;6:79-88

How to cite this URL:
Bolleddu R, Venkatesh S, Mangal AK, Varanasi S, Paria D, Prasad PV, Shiddamallayya N, Bandi V, Srikanth N. Botanical standardization, phytochemical analysis, and antioxidant studies of various fractions of Atibala [Abutilon indicum (L.) Sweet] leaves. J Drug Res Ayurvedic Sci [serial online] 2021 [cited 2022 Jun 29];6:79-88. Available from: http://www.jdrasccras.com/text.asp?2021/6/2/79/336035




  Introduction Top


Abutilon indicum (L.) Sweet belongs to the Malvaceae family, is a perennial shrub with a softly tomentose appearance, and grows up to 1–1.5 m in height. The plant is widely spread in India, Sri Lanka, tropical regions of America and Malaysia. In sub-Himalayan truants and hills, it is found up to 1200 m and densely found in hotter parts of India.[1],[2],[3] Its root is used in Ayurveda for the treatment of bleeding disorders (Raktapitta), Gout (Vatarakta), and excessive flow of urine (Meha). The dried roots of A. indicum are used in Ayurvedic formulations of Bala taila, Narayana taila, and Maha narayana taila.[4] Traditionally, its leaves are found to be beneficial for ulcers and as a poultice to painful parts of body.[5] Leaves decoction is used in tooth pain, tender gums, and for inflammation of bladder.[2],[6] Various scientific reports proved the significant pharmacological actions of A. indicum leaves. The petroleum ether and benzene extracts at the dose of 400 mg/kg were reported to possess significant analgesic property.[7] The aqueous, ethanolic, and chloroform extracts of A. indicum leaves were found to possess anti-inflammatory activity.[8] Alcoholic, aqueous extracts of leaves at a dose of 400 mg/kg were shown to exhibit significant hypoglycemic activity.[9] Ethanolic extract of leaves was found to exhibit a significant anticonvulsant effect.[10] Methanolic and aqueous extracts of A. indicum leaf reported remarkable anti-diarrheal activity.[11]A. indicum leaves methanolic extract shows significant antifungal activity (Trichophyton rubrum).[12] Methanol and petroleum ether extracts of A. indicum leaf have potent antibacterial activity (Pseudomonas aeruginosa, Staphylococcus aureus, and Klebsiella pneumonia).[13] The methanolic, petroleum ether, chloroform, ethyl acetate extracts of A. indicum leaf have remarkable cytotoxic activity.[14] Methanol extract of leaves showed significant antiulcer activity.[15] The crude leaf extracts of A. indicum were reported for its larvicidal activity.[16] Antioxidant is one of the important properties of medicinal plant for their therapeutic action, the antioxidant activity of various fractions of A. indicum was carried out along with botanical standardization to establish the quality parameters for A. indicum leaf.


  Materials and Methods Top


Chemicals

All the chemicals used were analytical grade. DPPH and phloroglucinol were obtained from Sigma-Aldrich. Ferric chloride, ascorbic acid, potassium ferricyanide, and butylated hydroxyl toluene were procured from S.D. Fine Chem Ltd. Iodine and potassium iodide were obtained from Merck.

Collection and identification

The fresh leaves were collected in the month of December 2015 from Suryapet (Dist.), Telangana state, India [Figure 1]. The plant was authenticated by Scientist and Taxonomist of the Botanical Survey of India, Hyderabad (Voucher specimen-GPRCP/AI/BR12/2015).
Figure 1: Morphology of Abutilon indicum (L.) Sweet leaves

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Macroscopy

Macroscopy was done by observing the fresh leaves under a simple microscope, and the color, size, texture, etc. were noted. Different macroscopic parameters such as shape, odor, and taste were noted.[17]

Anatomical studies

The freehand transverse sections of A. indicum fresh leaf are treated with chloral hydrate reagent, mounted in glycerin, and observed for histological characters using a digital microscope. By treating the section with phloroglucinol and concentrated hydrochloric acid (1:1), lignified tissues were determined.[18],[19]

Powder microscopy

The leaves were shade-dried, ground with a grinder, and sieved to obtain 60 mesh size powder. These shade-dried leaf powders are used for microscopic investigations. The powder was treated with chloral hydrate reagent, a little quantity of sample was taken onto a microscopic slide, 1–2 drops phloroglucinol reagent and iodine reagents were added separately, and a cover slip was placed above the sample. Then microscopic slides were sealed. The prepared slides were mounted and examined under a microscope. Tracing of characteristic structures and cell components was done.[20],[21]

Preparation of plant extracts

The shade-dried leaf powder of A. indicum (2 kg) was extracted with 80% of hydroalcohol by the maceration process for 8 days. The solvent was removed from the extract in a rotary evaporator. To 100 g of concentrated hydroalcoholic/mother extract, distilled water was added (500 mL) and fractionated with required quantity of petroleum ether, chloroform, ethyl acetate, and n-butanol.[22],[23]

Preliminary phytochemical screening

The mother extract and various fractions of A. indicum were tested for the detection of carbohydrates, proteins, alkaloids, glycosides, saponins, phenols, flavonoids, and steroids.[24]

Determination of total phenolic content

To the test solution (0.5 mL), 10% Folin–Ciocalteu reagent (5 mL) and 1 M of aqueous sodium carbonate (4 mL) were added. After 15 min, the absorbance was estimated at 765 nm. Standard curve of gallic acid (10–100 µg/mL) was plotted by absorbance vs. concentration.[25]

Determination of total flavonoid content

An aliquot of 1 mL of test sample is mixed with 3 mL of methanol, 0.2 mL of aluminum chloride (10%), 0.2 mL of potassium acetate (1 M), and 5.6 mL of distilled water. The reaction is allowed to remain at room temperature for 30 min. Reaction mixture absorbance was measured at 415 nm.[26]

Determination of diphenyl picrylhydrazyl radical scavenging activity

To 2 mL of DPPH solution (90 μM), different concentrations of 1 mL of test and standard (ascorbic acid) solutions were added. All the test tubes were incubated in dark condition for 1 h, and 1 mL of ethanol was added to each test tube. Absorbance of reaction mixture was measured at 517 nm. % Radical scavenging capacity was determined.[27]

%RSC = [Absorbance of Blank−Absorbance of Sample ÷ Absorbance of Blank] 100.

Determination of reducing power

To the test sample (1 mL) of different concentrations (100–1000 µg/mL), phosphate buffer (2.5 mL of 0.2 M, pH 6.6) and 1% potassium ferricyanide (2.5 mL) were mixed. The mixture was incubated for 20 min at 50°C. To this reaction mixture, 10% trichloroacetic acid (2.5 mL) was added and the mixture was centrifuged for 20 min at 3000 rpm. Top portion (2.5 mL) was mixed with water (2.5 mL) and 0.1% ferric chloride (0.5 mL), and absorbance was estimated at 700 nm. Butylated hydroxytoluene is used as the standard.[28],[29]


  Results Top


Macroscopy

The leaves are 8–10 cm long, green, base-cordate, stipulate, filiform, ovate shape, acuminate, pointed apex, reticulate venation, and toothed margin. Dorsal side is greener than the ventral side. It is hairy on both the surfaces [Figure 1].

Anatomical characters

Petiole

The transverse section of the petiole is almost round in shape, the outer layer of petiole is covered with plenty of stellate trichomes and few glandular trichomes, followed by cortical tissue which is formed of parenchymatous and collenchymatous cells [Figure 2]A, B. The middle region of the petiole consists of two pairs of 7–8 rows of vascular bundles surrounded by a group of rosette crystals which are scattered in the parenchymatous tissues.
Figure 2: Transverse section of Abutilon indicum (L.) Sweet leaf A. Outer region of petiole; B. Middle region of petiole; C. Midrib; D. Lamina St-Stellate trichomes; Cc-Collenchyma; Rc-Rosette crystals; Vb-Vascular bundles; Pc-Parenchyma cells; Ue-Upper epidermis; Vs- Vessels; Le-Lower epidermis; ­ Mgt-Multiseriate glandular trichomes; Mp-Mesophyll

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Midrib

The midrib consists of single row of rectangular-shaped upper epidermal cells with groups of stellate trichomes. Below the upper epidermis and above the lower epidermis, strips of 2–4-layered thick-walled collenchyma are present. The collateral vascular bundles appear at the center portion of the midrib and are composed of phloem and xylem cells. Rosette crystals are scattered in the phloem and xylem region. Lower epidermal cells are having greater number of groups of stellate trichomes than the upper epidermal cells. It consists of three different types of trichomes such as stellate type and uniseriate multicellular and unicellular stalk with unicellular head trichomes [Figure 2C].

Lamina

Upper epidermis is single-layered with more or less rectangular cells, covered with huge number of bunches of stellate trichomes, few uniseriate unicellular, multiseriate glandular trichomes. Mesophyll consists of bi-layered radially elongated palisade cells. It consists of rosette crystals, elongated vessels, and spongy parenchyma. Lower epidermis is having more bunches of stellate trichomes than the upper epidermis and 5–8 trichomes in each bunch [Figure 2D].

Powder microscopy

Under a microscope, A. indicum leaf powder shows numerous stellate trichomes, unicellular, multicellular glandular trichomes, anomocytic stomata, elongated sclerides with narrow lumen, lignified stone cells with large lumen, spiral vessels, long aseptate fibers, numerous prismatic, and rosette crystals. The results of powder microscopy are shown in [Figure 3] and [Figure 4].
Figure 3: Powder microscopy of Abutilon indicum (L.) Sweet leaf A. Leaf powder; B., C. Anomocytic stomata; D., E. Stellate trichomes; F. Unicellular glandular trichomes; G. Multicellular glandular trichome; H.,I. Stone cells; J., K. Elongated sclerides; L. Spiral vessel; M. Aseptate fiber; N. Rosette crystals; O. Prismatic crystals. (Scale bar=300 µm)

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Figure 4: Camera lucida drawing of powder microscopy of Abutilon indicum (L.) Sweet leaf A. Anomocytic stomata; B., C., D. Stellate trichomes; E. Unicellular glandular trichomes; F. Multicellular glandular trichome; G., H. Spiral vessel; I. Elongated sclerides; J. Stone cells; K. Aseptate fiber; L. Rosette crystals; M. Prismatic crystals.

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Extraction

The shade-dried A. indicum leaves were extracted with 80% aqueous ethyl alcohol by maceration at room temperature, and the yield of hydroalcoholic/mother extract is 130 g (6.5% w/w). The percentage yields of petroleum ether, chloroform, ethyl acetate, n-butanol, and remaining aqueous fraction of hydroalcoholic extract were 0%, 5.58%, 12.18%, 13.85%, and 62.17%.

Preliminary phytochemical screening

Preliminary phytochemical screening of the A. indicum extracts revealed the presence of flavonoids, steroids, and their glycosides, carbohydrates, phenols, proteins were present in [Table 1].
Table 1: Phytochemical analysis of various fractions of A. indicum (L.) Sweet leaves

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Estimation of total phenolic content

Phenols are the major plant compounds with antioxidant activity. The total phenolic content in plants usually correlates highly with the free-radical-scavenging activity. Among all fractions, ethyl acetate fraction (40 mg GAE/g) showed highest phenolic content, followed by butanol fraction (36 mg GAE/g). The total phenolic content is shown in [Table 2].
Table 2: Total phenolic and flavonoid content of all fractions

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Estimation of total flavonoid content

The total flavonoid content of mother extract and all the fractions were determined by the aluminum chloride assay using rutin as a standard. The total flavonoid content was found to be higher in ethyl acetate (30 mg RE/g) and chloroform (27 mg RE/g) fractions. [Table 2] represents the total flavonoid content.

DPPH radical scavenging activity

This assay is used widely as a primary test to provide information on the reactivity of test compound with a stable-free radical. DPPH in its radical form has purple color, gives strong absorption at 517 nm and when it is quenched by the extract, purple color disappears in the presence of a substance, which can donate hydrogen depending on the antioxidant activity. Different extracts of A. indicum exhibited remarkable DPPH radical scavenging activity, as shown in [Figure 5]. Ethyl acetate (65 µg/mL), butanol (95 µg/mL) fractions DPPH radical scavenging capacity is higher than all other fractions. The decreasing order of DPPH radical scavenging activity is ascorbic acid (IC50-1.3 µg/mL)>AILE (IC50-65 µg/mL) >AILB (IC50-95 µg/mL)>AILC (IC50-350 µg/mL) >AILA (IC50-350 µg/mL) >AILM (IC50-700 µg/mL).
Figure 5: Antioxidant studies of A. indicum (L.) Sweet leaf

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Determination of reducing power

The measurement of reductive ability is a significant indicator for the determination of potential antioxidant activity. The reducing properties are generally associated with the presence of reductones, which cause breakdown of chain reactions by donating hydrogen atoms. Reductones also react with certain precursors of peroxide, thus preventing formation of the latter. Increasing of absorbance values indicates increased reducing power activity. Standard butylated hydroxytoluene (BHT) has shown good reductive capabilities in the concentration range of 1–10 µg/mL. Hydroalcoholic/mother extract has shown good reductive capabilities in the concentration range of 10–100 µg/mL. Remaining fractions have shown good reductive capabilities in the concentration range of 100–1000 µg/mL. The decreasing order of reductive capabilities were BHT >AILM > AILE > AILC > AILB > AILA. Reducing power of test and standard drugs was shown in [Figure 5].


  Discussion Top


The present investigation was carried out to evaluate the macro-microscopical, phytochemical profile, and antioxidant potential of A. indicum leaves by using different in-vitro models. Botanical standardization is having a key role in establishing the proper identity of medicinal plants and also plays a main role in guaranteeing the phytochemical quality and clinical efficacy. Macro- and microscopic evaluations (histological studies, powder microscopy) are the main stay of botanical standardization which can identify the adulterants and substituents.[30] Transverse section of the fresh leaf of A. indicum showed the presence of stellate trichomes and glandular trichomes; rosette crystals in petiole and midrib consist of three different types of trichomes such as stellate type and uniseriate multicellular and unicellular stalk with unicellular head trichomes. Powder microscopy shows diagnostic characters like anomocytic stomata, elongated sclerides, lignified stone cells, and spiral vessels. These anatomical and powder microscopical studies can be considered as reference standard for future studies on A. indicum leaves.

The A. indicum leaves were shade-dried, ground, and extracted with 80% aqueous ethyl alcohol by cold maceration and fractionated with various organic solvents, viz., petroleum ether, chloroform, ethyl acetate, and n-butanol for the separation of polar and non-polar phytoconstituents. These fractions were further subjected to qualitative phytochemical investigations and screened for antioxidant studies. The preliminary phytochemical screening (test tube reactions) of A. indicum revealed the presence of phenols, flavonoids, proteins, and carbohydrates in all fractions.

The antioxidative system protects the organism against the reactive oxygen species (ROS)-induced oxidative damage. Natural substances gain popularity as antioxidants compared with synthetic antioxidant.[31] An attempt was made to assess the antioxidant capabilities of all fractions of A. indicum leaves. In DPPH radical scavenging activity, ethyl acetate extract reported superior activity among all extracts. The concentration of ethyl acetate extract resulting in 50% inhibition of free radical was 65 µg/mL, followed by butanol extract, chloroform, leftover aqueous extract, and hydroalcoholic extract. The significant decrease in concentration of DPPH radical is due to the scavenging ability of test extracts. In reducing power assay, decrease in absorbance is indicated as an increase in reducing power. Increased reducing power is associated with increased antioxidant potential. In this method, the presence of antioxidants in the extract would result in reducing of Fe³+ to Fe²+ by donating an electron. Amount of Fe²+ complexes is estimated by measuring the formation of blue color at 700 nm. All fractions of A. indicum leaf show positive results in this reducing power assay. The hydroalcoholic extract exhibited superior activity than all other fractions of A. indicum leaf.

The total phenolic and flavonoid contents of all fractions were determined. The literature reveals that phenols and flavonoids are known for their antioxidant properties.[32] The highest concentration of total phenolic and flavonoid content was found to be 40 mg GAE/gm and 30 mg RE/g, respectively, in ethyl acetate extract. The observed superior antioxidant activity may be attributed to the presence of these compounds.


  Conclusion Top


The present investigation established the qualitative diagnostic features of leaves of A. indicum through anatomical, powder microscopical, phytochemical, phenolic content, and flavonoid content studies. Phytochemical analysis revealed that all the fractions are rich sources of carbohydrates, alkaloids, steroids, saponins, phenols, and flavonoids. Among all fractions, ethyl acetate (40 mg GAE/g) followed by butanol fraction (36 mg GAE/g) reported higher total phenolic content. Similarly, ethyl acetate (30 mg RE/g) is followed by chloroform fraction (27 mg RE/g); total flavonoid contents were more among all fractions. In DPPH radical scavenging activity, ethyl acetate (IC50-65 µg/mL) and butanol fraction (IC50-95 µg/mL) reported significantly greater scavenging activity, whereas in reducing power assay, hydroalcoholic extract followed by ethyl acetate fraction exhibited greater reducing power. More phenolic and flavonoid contents and higher level of antioxidant activity was observed in ethyl acetate fraction. Hence, ethyl acetate fraction of A. indicum can be considered as a potential source of natural antioxidant.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2]



 

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