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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 15  |  Issue : 4  |  Page : 280-286

Macromicroscopical, physicophytochemical, and high-performance thin layer chromatography-based diagnostics of an antihypertensive drug "sarpgandha" resolving adulterant/substitute


Pharmacognosy Division, CSIR- National Botanical Research Institute, Rana Pratap Marg, Lucknow, India

Date of Submission28-Nov-2020
Date of Decision11-Feb-2021
Date of Acceptance25-Mar-2021
Date of Web Publication16-Dec-2021

Correspondence Address:
Saba Irshad
Division of Pharmacognosy, CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow - 226 001, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/joa.joa_299_20

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  Abstract 


Introduction: "Sarpgandha" or "Asrol" is the root of Rauvolfia serpentina (L.) Benth Ex. Kurz., which belongs to the family Apocynaceae. Conventionally, it is used in the treatment of several body ailments, viz., abdominal pain, circulatory disorders, dysentery, gastrointestinal disease, hypertension, mental illness, malaria, and snakebite, and as a uterine stimulant; however, its monoterpenoid indole alkaloid reserpine extracted from the root is used for the treatment of hypertension. The problem reported that, occasionally, stem or aerial part of the same plant adulterated along with the root. Therefore, the aim of the present study is to carry out pharmacognostic evaluation of "Sarpgandha" and its adulterant/substitute for quality control. Methods: Macromicroscopy, physicochemical parameters, and high-performance thin layer chromatography (HPTLC) profiling have been done. Results: The study showed that the root has dark vertical ridges on the surface whereas stem has no ridges but mark of nodes and internodes. Transverse section of the root shows highly sutured outer lining while that of the stem shows smooth lining. Powder microscopy showed cork cells and starch grains in the root while single and group of stone cells in the stem. All the studied physicochemical parameters present higher level in the root powder, except total moisture content. HPTLC densitometric profile shows alkaloid reserpine at retention factor 0.49 under UV254 and UV366 nm in the solvent system toluene: ethyl acetate: diethylamine (7:2:0.5) in the methanolic extract of the root. Conclusion: Quality control markers of "Sarpgandha" would be available for authentication of commercial samples.

Keywords: Adulteration, hypertension, quality control, Rauvolfia serpentina, Sarpgandha


How to cite this article:
Irshad S, Khatoon S. Macromicroscopical, physicophytochemical, and high-performance thin layer chromatography-based diagnostics of an antihypertensive drug "sarpgandha" resolving adulterant/substitute. J Ayurveda 2021;15:280-6

How to cite this URL:
Irshad S, Khatoon S. Macromicroscopical, physicophytochemical, and high-performance thin layer chromatography-based diagnostics of an antihypertensive drug "sarpgandha" resolving adulterant/substitute. J Ayurveda [serial online] 2021 [cited 2022 Jan 27];15:280-6. Available from: http://www.journayu.in/text.asp?2021/15/4/280/332605




  Introduction Top


Quality drug is the core to any therapeutic preparation, which is determined from the beginning of plant material collection and end up to drug delivery.[1],[2] "Sarpgandha" or "Asrol" is the root of Rauvolfia serpentina L. Benth., belongs to the family Apocynaceae, and is an important medicinal herb prescribed in ancient Ayurvedic literature to cure heart and brain disease. It is grown in the upper Gangetic plain.[3] Occasionally, aerial part of the same plant mixed with the root is not prescribed in reference Ayurvedic text or may cease the quality of drug.[4] Conventionally, R. serpentina is being used in the treatment of several ailments, viz., abdominal pain, circulatory disorders, hypertension, mental illness, malaria, and snakebite.[5] In 1949, this plant was first time reported for treating high blood pressure.[6]

The major phytochemicals reported from this plant are alkaloid from monoterpenoid indole family, viz., ajmaline, ajmalimine, ajmalicine, deserpidine, indobine, indobinine, reserpine, reserpiline, rescinnamine, rescinnamidine, serpentine, serpentinine, and yohimbine. However, other phytochemicals such as carbohydrate, flavonoids, and tannins are also reported from this plant.[6] Pharmacologically, R. serpentina has been reported for antihypertensive, antimicrobial, antifungal, anti-inflammatory, antiproliferative, antidiuretic, and anticholenergic activity and also found profound activity against drug-resistant tumor cell.[7]

The pharmacognostic evaluation of medicinal plant is highly required for the development of quality control markers (macromicroscopical, physicophytochemical, and thin layer chromatography) for proper identification of potential plant species and plant part for quality drug. In the above context, the aim of the present study to evaluate the comparative macromicroscopy, physicochemical parameters, and high-performance thin layer chromatography (HPTLC) profiling of the root and aerial part of R. serpentina (L.) Benth. for the development of quality control markers.


  Materials and Methods Top


Collection of plant material

The selected plant species of Rauvolfia was collected from CSIR-NBRI garden in April 2018. Herbarium specimen was prepared as per the standard herbarium procedure[8] and deposited in the herbarium of CSIR-National Botanical Research Institute, Lucknow wide accession number LWG-104702. Institutional MS no. 'CSIR-NBRI_ MS/2020/10/09' allotted by the ethical committee CSIR-NBRI, Lucknow.

Macromicroscopical studies

Macroscopy of the root and stem part of Rauvolfia species was done with the help of flora.[3] Microscopy and powder analysis were done as per standard method.[9],[10]

Physicophytochemical parameters

Total moisture content, total ash, acid-insoluble ash, water-soluble extractive, and alcohol-soluble extractive were calculated as per the Pharmacopoeial method.[11] Total sugar, total starch content according to standard method,[12],[13] and total alkaloid content were calculated.[14]

Preparation of sample for high-performance thin layer chromatography

For HPTLC studies, plants were air-dried at room temperature in the shade and were used for solvent extraction. The root and aerial part of R. serpentina grounded to a coarse powder and placed in appropriately sized volumetric flasks. 100 mL methanol was added to 10 g of powder of each plant shaken on shaker for 2 h, kept at rest overnight, and filtered thereafter. The procedure was repeated thrice with methanol (100 mL) at room temperature (25°C ± 2°C). The methanolic extracts were filtered through Whatmann No. 1 filter paper and combined. The combined extracts were concentrated under reduced pressure using Rotavapor R-114 (Buchi) at a temperature of 45°C and freeze-dried in Freeze Dry System/Freezone 4.5 (Labconco).

Accurately weighed 10 mg of the extract was dissolved in 1 mL methanol and filtered through a 0.45 μm filter membrane; the filtrate was used as sample solution. 1 mg of standard reserpine was dissolved in 10 mL methanol to get 0.1 mg/mL solution of standard markers.

High-performance thin layer chromatography

HPTLC was performed on 10 cm × 10 cm silica TLC aluminum sheet, coated with 0.2 mm layer of silica gel containing UV 254 fluorescent indicator (S D Fine Chemicals, India). Samples (15 μL) and standards (10 μL) were applied to the plates by means of a Camag (Switzerland) ATS4 sample applicator. The plates were developed distance of 8 cm from the lower edge of the plate with 10 mL toluene–ethyl acetate–diethyl amine (7:2:0.6 v/v/v) as mobile phase, in a Camag twin-trough chamber, previously saturated with mobile phase vapor for 30 min at 25°C ± 2°C. After removal from the chamber, plates were completely dried in air at room temperature (25°C ± 2°C) and documented under UV 254 nm and UV 366 nm through Visualize 2 documentation system. Densitometric scanning was performed on a Scanner4 with Server desktop-f16 g2j2, version 2.3.17006.1 software Camag (Switzerland) at a wavelength of 250 nm for reserpine.[15]

Statistical analysis

All the physicophytochemical experiments were performed thrice and the results have been given as mean value with ± standard deviation (SD).


  Results Top


Macroscopy

Root – cylindrical slightly tapering, hard, curved, 20–50 cm in length, 1.5–3 cm in diameter, scars of rootlets present; root surface is dull brown with coarse vertical and horizontal ridges, along with the inner bark at pale yellow in color [Figure 1]A. Stem appears light brown with rough surface, along with marks of nodes and internodes [Figure 1]A. Leaves – in the whorl of 3, lanceolate, 10–15 cm × 3–5 cm in size, glabrous, bright green above, pale green beneath, apex acute to shortly acuminate, base attenuate, main nerves 8–10 pairs, petiole 1–2 cm [Figure 1]A. Flower – white often tinged with pink, many flowers irregular corymbose cymes, drupes connate except at top, 0.6–1 cm in claim [Figure 1]A. Fruit appears purplish and black when ripe [Figure 1]A.
Figure 1: Macromicroscopy of Rauvolfia serpentina. (A) Macroscopy – (a) whole plant, (b) root, (c) stem, (d) leaf, (e) flower, (f) fruit. (B) Microscopy – (c) transverse section root, (b) transverse section stem, (c) transverse section leaf, (d) transverse section petiole. Ck: Cork, ct: Cortex, cu: Cuticle, gt: Ground tissue, lepi: Lower epidermis, mr: Medullary rays, pal: Palisade tissue, pi: Pith, sc: Stone cells, scph: Secondary phloem, sg: Starch grains, sp: Spongy parenchyma, uepi: Upper epidermis, xy: Xylem

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Microscopy

Transverse section (TS) of the root have externally several layers of polygonal to tabular cork cells forming two types of band alternating with each other. One type of band consists of 1–5 layers of narrow cells with suberized but unlignified walls followed by a band of 1–2 layers of broad cells with suberized and lignified walls. The radial walls of the broad cells tend to break and as a consequence the cork frequently peels off in layers. The cork is followed by a layer of phellogen cells, and phelloderm consists of about 12 layers of cells having smaller rectangular and tabular cells. Few cells of phelloderm contain yellowish granular content. Medullary rays running radially and are 2–4 cells wide. The cells of medullary rays are rounded rectangular or ovoid with small intercellular spaces. The sieve tissue consists of sieve tubes, companion cells, and phloem parenchyma. Cells of phloem parenchyma contain starch grains. Xylem contains strongly lignified cells. All the cells of xylem parenchyma and medullary rays contain abundant starch grains [Figure 1]B.

TS of the stem showed that the outermost layer of cork comprises 5–6 layers of rectangular cells filled with stone cells, followed by cortex consist of pentagonal cells with stone cells; next to cortex, pericyclic fibers were present followed by secondary phloem, and xylem region consists of pitted xylem vessels, xylem trachids, and uniseriate medullary rays; the innermost pith consists of single and group of stone cells [Figure 1]B.

TS of the leaf shows that it consists of midrib and laminar region. Midrib region is somewhat irregular semicircular in shape with arch-shaped vascular region consisting of xylem and phloem surrounded by ground tissue. In the laminar region on the upper surface, upper epidermis followed by a layer of palisade tissue then spongy parenchyma; on the lower surface, lower epidermis present [Figure 1]B.

Powder microscopy

Root: Yellow rough textured powder with pungent odor and bitter taste. Under microscope, powder showed single and group of starch grains, prismatic crystals of calcium of oxalate, cork cells, part of medullary rays, pitted xylem vessels with perforations, pitted trachids, resin containing cells, lignified xylem fibers, etc., [Figure 2]A. Stem: Dull brown coarse powder, slight odor, bitter taste. Microscopic analysis showed single and group of stone cells, parenchymatous cells, highly pitted xylem vessel, pitted xylem trachids, medullary rays, etc., [Figure 2]B.
Figure 2: Powder microscopy of the root and aerial part of Rauvolfia serpentina. (A) Root, (B) Aerial part. (a) Cork cells, (b) hexagonal parenchymatous cells, (c) starch grain containing xylem trachids, (d) resin containing mass of cells, (e) pitted xylem vessels, (f) prismatic crystals of calcium oxalate, (g) palisade tissue, (h) elongated parenchymatous cells, (i) stone cells, (j) xylem vessel with spiral thickening, (k) epidermal cells with stomata, (l) cortical cells

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Physicophytochemical analysis

Physicochemical parameters such as total moisture content, total ash, acid-insoluble ash, water-soluble and alcohol-soluble extractive, total sugar, total starch, and total alkaloid were calculated and the results are shown in [Figure 3].
Figure 3: Physiophytochemical parameters of root and aerial part of Rauvolfia serpentina (a and b). TMC: Total moisture content, TA: Total ash, AIA: Acid-insoluble ash, WSE: Water-soluble extractive, ASE: Alcohol-soluble extractive, TSHC: Total sugar content, TSC: Total starch content, TAC: Total alkaloidal content

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High-performance thin layer chromatography

HPTLC profiling of methanolic extract of R. serpentina root and stem was done with one bioactive monoterpene indole alkaloid marker reserpine. Toluene–ethyl acetate–diethyl amine (7:2:0.5 v/v/v) was used as mobile phase and profiling has been done under UV254 nm and UV366 nm which is shown in [Figure 4]. The retention factor values of substance bands are presented in [Table 1]. Scanning has been done at 250 nm and densitometry chromatogram of methanolic extract of root and aerial part of R. serpentina is depicted in [Figure 5].
Figure 4: High-performance thin layer chromatography profile of methanolic extract of root and aerial part of Rauvolfia serpentina along with marker reserpine in the solvent system for alkaloid (toluene-ethyl acetate-diethyl amine, 7:2:0.5). (a) Under UV245 nm, (b) under UV366 nm. (1) Aerial part, (2) root, (r) reserpine

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Figure 5: High-performance thin layer chromatography densitometry chromatogram of (a) standard reserpine, (b) aerial part, and (c) root extract of Rauvolfia serpentina scan at 250 nm

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Table 1: Comparative Rf details of root and aerial part of Rauvolfia serpentina along with reserpine in the solvent system for alkaloid (toluene–ethyl acetate–diethyl amine, 7:2:0.5)

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  Discussion Top


Substitution in herbal drug is required in the present scenario as a list of red data book increase up to 300 medicinal plants. However, adulteration is an erroneous exercise of mixing/replacing the genuine raw material with other substances partially or fully, which is inferior in therapeutic and chemical properties or no desired bioactivity, due to lower cost or unavailability of original one for the personal interest of profit gain. The concept of substitution focused on the pharmacological activity of herbal preparation rather than morphology and anatomical structure.[16] To resolve the problem of adulteration/substitution, the WHO has set several parameters for the standardization of herbal drug, viz., (a) botanical characters, sensory evaluation, foreign organic matter, microscopic, histological, histochemical assessment, quantitative measurements, and (b) physical and chemical identity, fingerprints chromatography, ash values, extractive values, moisture content and alkaloids tests, quantitative estimation protocols.[11],[17] In pharmacopoeias also, the Rf values of general TLC/HPTLC profiles have been included as minimum requirement of quality standards of plant-based single drug or formulations.[2]

In the present study, comparative macroscopy of root and stem part of R. serpentina showed that although there are some similarities in two studied parts, but some differentiating characters are also present which are noticeable, such as root is curved, tapering with dark vertical ridges present on the surface with inner side yellow color whereas stem has no ridges on the surface but mark of nodes and internodes present [Figure 1]A. Microscopical studies showed TS. root with highly sutured outer lining while TS. stem have smooth outer lining. Layer of cork cells present in root while absent in stem. Cortical region broad in stem but compressed in root. High amount of starch grains present in cortical region in root while absent in stem. Single and group of stone cells present in cortical and pith region of stem while absent in root [Figure 1]B. Powder microscopy of two studied parts of R. serpentina showed cork cells and starch grains in the powder of root while single and group of stone cells are present in the powder of stem [Figure 2].

Air-dried material was used for quantitative determination of physicophytochemical values. Extractive values (alcohol and water soluble), ash values (total and acid insoluble ash), total sugar and starch content, and total alkaloid content were repeated for six times, and mean values ± SD are presented in [Figure 3]. Data showed that aerial part has comparatively more percentage of moisture content (12.48 ± 0.36) than root (10.48 ± 0.45), and it is significantly involved in crude drug storage and shelf-life as well as knowledge of suitable condition for growth of microorganism.[17] Roots have more quantity total and acid-insoluble ash (5.14 ± 0.12 and 1.28 ± 0.05, respectively) than aerial part (3.42 ± 0.22 and 0.96 ± 0.12, respectively); thus, the roots showed the higher level of inorganic matter (carbonates and silicates) while aerial part contains the minimum inorganic mineral, it is important for the quality of herbal drug, and samples showed very less amount of acid-insoluble ash value that may affect the amount of the component absorbed in the gastrointestinal canal when taken orally.[15],[18] Water- and alcohol-soluble extractive value that found also higher in the root (21.45 ± 0.05 and 10.85 ± 0.50, respectively) than aerial part (13.45 ± 0.17 and 8.24 ± 0.27, respectively) shows both polar and nonpolar compounds present in good amount in root part.[11],[15],[17],[18] Starch and sugar percentage also high in root comparative to aerial part [Figure 3], evident from microscopical study that large amount of starch grain contained in phloem and xylem regions. It is pharmaceutically significant in storage and formulation process for a particular disease like diabetes.[15],[19] Amount of alkaloid is higher in root (2.56±0.20) than aerial part (1.58±0.23). Physicochemical data support the medicinal importance of root part comparative to aerial part.

In the HPTLC profile of methanolic extract of root and stem part of R. serpentina[5] band of reserpine present at Rf 0.49 under both UV254 and UV366nm in methanolic extract of root. However, reserpine was absent in the extract stem part [Figure 4]. HPTLC densitometry chromatogram scanned at 250 nm clearly showed the peak of reserpine only in root extract of R. serpentina [Figure 5]. In the present study, details of the Rf values of each band given in [Table 1] can be used to identify compounds due to their uniqueness and give the idea of individual compounds present in studied sample extract, which may thus be used as a quality control profile for this drug.[11],[15],[17]


  Conclusion Top


The above study successfully established the macromicroscopical, physicophytochemical parameters, and HPTLC of the root and aerial part of important drug "Sarpgandha" and its adulterant/substitute for quality control point of view. The developed data may be used as a reference tool for proper confirmation of right plant material and monitoring of batch-to-batch consistency of finished herbal drug for pharmaceutical industries and also the reference material for further study.

Acknowledgment

The authors are thankful to Director CSIR-National Botanical Research Institute, Lucknow, for providing lab facility for this research work. First author is also thankful to Council of Science and Technology, UP, for financial support.

Financial support and sponsorship

CST-UP, Young Scientist Fellowship with grant letter no. CST/8272, supported the study.

Conflicts of interest

There are no conflicts of interest.





 
  References Top

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Chawla R, Thakur P, Chowdhry A, Jaiswal S, Sharma A, Goel Rajeev, et al. Evidence based herbal drug standardization approach in coping with challenges of holistic management of diabetes: A dreadful lifestyle disorder of 21st century. J Diabe Metab Disorders 2013;12:35.  Back to cited text no. 19
    


    Figures

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

  [Table 1]



 

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