|
 
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| ORIGINAL ARTICLE |
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| Year : 2023 | Volume
: 8
| Issue : 3 | Page : 250-261 |
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Pharmacognostic study and high-performance liquid chromatography finger print profile of herbs of Hridyadashemani
Harini Aswatha Narayan Rao1, Veena Mullur Siddalingaiah2, Lalitha B Ramachandraiah3
1 Government Ayurveda Medical College, Bengaluru, India; Department of Dravyaguna, Sri Dharmasthala Manjunatheshwara College of Ayurveda & Hospital, Hassan, India
2 Department of Dravyaguna, Government Ayurveda Medical College, Bengaluru, India
3 Department of Dravyaguna, K T G Ayurveda Medical College, Bangalore, Karnataka, India
| Date of Submission | 03-Sep-2022 |
| Date of Acceptance | 03-Feb-2023 |
| Date of Web Publication | 16-Aug-2023 |
Correspondence Address:
Dr. Harini Aswatha Narayan Rao
Department of Dravyaguna, Sri Dharmasthala Manjunatheshwara College of Ayurveda and Hospital, Hassan 573201, Karnataka
India
 Source of Support: None, Conflict of Interest: None
DOI: 10.4103/jdras.jdras_138_22
BACKGROUND: In Ayurveda, medicinal plants have been organized into various categories based on several parameters. One such classification is based on actions, wherein the therapeutic agents have been categorized into 50 groups. Among these, Hridyadashemani is one that includes the fruits of Amra, Amrataka, Lakucha, Karamarda, Vrukshamla, Amlavetasa, Kuvala, Badara, Dadima, and Matulunga. The objective of this study was to identify and develop preliminary analytical standards for the ten herbs of Hridyadashemani. METHODS: The herbs were collected from their natural habitat and subjected to macroscopic and microscopic evaluation as per standard procedures. The analytical study that includes foreign matter (%w/w), total ash (%w/w), acid insoluble ash (%w/w), alcohol-soluble extractive (%w/w), and water-soluble extractive (% w/w) of the herbs were conducted as per standard pharmacopeial guidelines. Preliminary phytochemical evaluation and high-performance liquid chromatography (HPLC) analysis of the herbs for the presence of quercetin and gallic acid were carried out. RESULTS: The macroscopic and microscopic features of the fresh and dried fruits were documented and compared to published literature. The analytical standards were recorded and compared to pharmacopeial standards and published literature. The herbs were found to contain various quantities of quercetin and gallic acid. These phytochemicals may have a potential role in cardiovascular diseases. CONCLUSION: The preliminary analytical findings include HPLC finger print profile which would serve as a monograph for Hridyadashemani. Further studies may be taken up to analyze the use of these herbs in various cardiovascular diseases. Keywords: Ayurveda, cardioprotection, gallic acid, Hridyadashemani, pharmacognosy, quercetin
How to cite this article:
Narayan Rao HA, Siddalingaiah VM, Ramachandraiah LB. Pharmacognostic study and high-performance liquid chromatography finger print profile of herbs of Hridyadashemani. J Drug Res Ayurvedic Sci 2023;8:250-61 |
How to cite this URL:
Narayan Rao HA, Siddalingaiah VM, Ramachandraiah LB. Pharmacognostic study and high-performance liquid chromatography finger print profile of herbs of Hridyadashemani. J Drug Res Ayurvedic Sci [serial online] 2023 [cited 2023 Sep 23];8:250-61. Available from: http://www.jdrasccras.com/text.asp?2023/8/3/250/383695 |
| Introduction | |  |
Medicinal plants have been classified under various headings for better understanding and application in therapeutics. Hridyadashemani (Hridyamahakashaya) are 10 herbs delineated to be conducive to the heart, which include Amra, Amrataka, Lakucha, Karamarda, Vrukshamla, Amlavetasa, Kuvala, Badara, Dadima, and Matulunga.[1] Accurate identification and authentication of herbal medicines are essential for their safety and efficacy. To achieve this, morphological, microscopic, and chemical identification are the primary techniques adopted.[2] Botanical reference standard (BRS) regarding its botanical identity and plant part is necessary to relate to specific therapeutic effect. The quality and purity of herbs play a pivotal role in its efficacy; hence, the World Health Organization (WHO) has emphasized the need to ensure the quality of medicinal plant products by using modern control techniques and applying suitable standards.[3] Standardization assures that herbs are reliable in identity, quality, purity, safety, and efficacy.[4] As per the authors’ knowledge, there are no comprehensive standards on Hridyadashemani and hence this study was conducted with the objective of developing a preliminary pharmacognostic evaluation with high-performance liquid chromatography (HPLC) finger print profile for the ten herbs of Hridyadashemani.
| Materials and Methods | | |
Plant collection and authentication
The fruits of Amra (Mangifera indica L.), Amrataka (Spondias pinnata (L.f.) Kurz), Lakucha (Artocarpus lacucha Roxb. ex Buch.-Ham., Karamarda (Carissa carandas L.), Vrukshamla (Garcinia indica (Thouars) Choisy), Amlavetasa (Garcinia pedunculata Roxb. ex Buch.-Ham.), Kuvala (Ziziphus jujuba Mill.), Badara (Z. jujuba Lam.), Dadima (Punica granatum L.), and Matulunga (Citrus medica L.) were collected from their natural habitat [Figure 1]A–J. The collected samples were authenticated by scientists at ICMR-National Institute of Traditional Medicine, Belagavi, and herbarium of these plants is maintained in the laboratory (Voucher specimen No.: RMRC-1619-1627). | Figure 1: Macroscopic features of the herbs of Hrdyadashemani. (A) Fresh and dried fruits of Amra (Mangifera indica Linn.). (B) Fresh and dried fruits of Amrataka (Spondias pinnata (Linn.f.) Kurz). (C) Fresh and dried fruits of Lakucha (Artocarpus lakoocha Roxb.). (D) Fresh and dried fruits of Karamarda (Carissa carandas Linn. Mant.). (E) Fresh and dried fruits of Vrukshamla (Garcinia indica Chois.). (F) Fresh and dried fruits of Amlavetasa (Garcinia pedunculata Roxb.ex Buch.-Ham). (G) Fresh and dried fruits of Kuvala (Ziziphus jujuba Mill.). (H) Fresh and dried fruits of Badara (Ziziphus jujuba Lam.). (I) Fresh and dried fruits of Dadima (Punica granatum L.). (J) Fresh and dried fruits of Matulunga (Citrus medica L.)
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Plant processing
The collected fruits were cleaned, whole fruits were chopped, except for Amra, Vrukshamla, Amlavetasa, Kuvala, and Badara, whose endocarp with seeds was removed before chopping into smaller pieces and dried under shade [Figure 1]A–J. A portion of the dried herbs were pounded into coarse, fine powder and stored in containers for pharmacognostic studies. Remaining dried herbs were stored in air tight containers for further study.
Pharmacognostic study
The dried herbs were subjected to the following evaluation as per the guidelines of Ayurvedic Pharmacopeia of India (API)[5] and was conducted at the Department of Dravyaguna.
Macroscopic and sensory evaluation
Macroscopic details of the fresh and dried herbs were observed with the naked eye or with a magnifying lens. The general condition of the herb, size, shape, surface, and texture was noted. Sensory characteristics such as color, odor, and taste were recorded.
Powder microscopy
A pinch of fine powder was put on the slide, treated with chloral hydrate, phloroglucinol with HCl, iodine solution mounted in glycerine, and powder characters observed under a compound microscope. The features were photographed using camera under bright-field light.
Physicochemical evaluation
The physicochemical evaluation includes tests as per API for herbs. This included foreign matter (%w/w), total ash (%w/w), acid-insoluble ash (%w/w), alcohol-soluble extractive (%w/w), and water-soluble extractive (% w/w). All the tests were performed in triplicates, and data were presented as mean ± standard deviation.
Qualitative phytochemical evaluation
The aqueous and alcoholic extracts of all ten dried herbs were screened for phytoconstituents using standard reagents and methodology.[6]
High-performance liquid chromatography-quantification of gallic acid and quercetin
About 10 g of each sample was dissolved in 20.0 mL of methanol, kept overnight, filtered, removed solvent under reduced pressure, and dissolved in ethanol to prepare 10 mg/mL of each of the extract. Gallic acid and quercetin (100 µg/mL each were prepared from the stock of 1 mg/mL by dissolving in methanol). Samples were injected automatically into a Shimadzhu LC-Prominence 20AT HPLC system. Absorption spectra for the main peaks were recorded at 275 nm. The HPLC system was equipped with a C18 column 250 nm × 4.6 mm and a spherical particle size of 5 µ, which was kept at 25°C. The mobile phase comprises of HPLC grade acetonitrile (60%) and HPLC water (40%) at a flow rate of 1 mL/min. The injection volume was 10 µL.
| Observations and Results | | |
The macroscopic and sensory characters of the fresh and dried herbs are tabulated in [Table 1] and [Table 2], respectively [Figure 1]. The prominent powder characters of the ten herbs are tabulated in [Table 3] and [Figure 2]. The physicochemical constants of the ten herbs are tabulated and compared with standard monographs [Table 4]. Standard monographs are not available for Amra, Lakucha, and Kuvala and hence the results obtained from this study could serve as a standard. In the qualitative phytochemical evaluation, the major chemicals detected were primary metabolites such as carbohydrates, proteins and secondary metabolites such as saponins, alkaloids, and tannins [Table 5]. The HPLC analysis revealed various amounts of gallic acid and quercetin [Table 6] and [Table 7]. | Table 1: Macroscopic and sensory characters of fresh herbs of Hridyadashemani
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 | Table 2: Macroscopic and sensory characters of dried herbs of Hridyadashemani
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 | Figure 2: Powder microscopy of the herbs of Hrdyadashemani. (A) Microscopic features of Amra (Mangifera indica L.). (B) Microscopic features of Amrataka (Spondias mangifera Willd.). (C) Microscopic features of Lakucha (Artocarpus lakoocha Wall.exRoxb). (D) Microscopic features of Karamarda (Carissa carandus L.). (E) Microscopic features of Vrukshamla (Garcinia indica Choisy). (F) Microscopic features of Amlavetasa (Garcinia pedunculata Roxb.ex Buch.- Ham). (G) Microscopic features of Kuvala (Ziziphus jujuba Mill.). (H) Microscopic features of Badara (Zizyphus jujuba Lam.). (I) Microscopic features of Dadima (Punica granatum L.). (J) Microscopic features of Matulunga (Citrus medica L.)
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 | Table 4: Showing the physicochemical constants of herbs of Hridyadashemani
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 | Table 5: Showing the preliminary phytochemicals of herbs of Hridyadashemani
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| Discussion | | |
Identification of the herbs of Hridyadashemani
The herbs of Hridyadashemani are 10 in number, which includes Amra, Amrataka, Lakucha, Karamarda, Vrukshamla, Amlavetasa, Kuvala, Badara, Dadima, and Matulunga.[1] The used part of these ten herbs is stated to be fruits,[7] which confirms their BRS. These herbs have been botanically identified as Amra, the fruits of M. indica L.,[8]Amrataka as the fruits of S. pinnata [(L.f.) Kurz],[9]Karamarda as the fruits of C. carandas L.,[10]Badara, as the fruits of Z. jujuba Lam.,[11]Dadima as the fruits of P. granatum L.,[12] and Matulunga as the fruits of C. medica L.[13] The plant Lakucha has been identified as A. lacucha Roxb. ex Buch.-Ham.,[14] and the fruits of the plant were taken for the this study. Vrukshamla is identified as Garcinia indica Chois.,[15] and the fruits of the same were collected for the study.
Kuvala refers to a variety of Badara with larger sized fruits.[16] About 18–20 Ziziphus species are found in the Indian subcontinent, of which six are recognized as economically important species.[17]Ziziphus sativa Gaertn. fruits are oblong or narrowly ovoid, 2–3.5 cm, 1.5–2 cm in diameter,[18] which are bigger than the fruits of Z. jujuba Lam., which are oblong or globose, 1–1.2 cm, ca. 1 cm in diameter,[19] and hence Kuvala may be considered and have been identified as Ziziphus sativa Gaertn.[20]Ziziphus sativa Gaertn. is regarded as a synonym for Z. jujuba Mill.,[21] and hence Z. jujuba Mill. was taken as Kuvala in this study. Though various plant sources are used in Amlavetasa, G. pedunculata Roxb.ex Buch.-Ham has been identified and accepted as the botanical source,[22],[23] which has been described under the name Vruntamlaphala in API.[24] Hence, G. pedunculata Roxb.ex Buch.-Ham fruits were collected for the study.
Pharmacognostic evaluation of the individual herbs of Hridyadashemani
Assessment to determine the pharmacognostic analytical standards is the preliminary step towards establishing identity and the degree of its purity.[4] Standardization is a tool in the quality control process, and published monographs are the most practical approach towards achieving quality control. When standard monographs are unavailable, development and validation of analytical procedures should follow the pharmacopeial definitions of identity, quality, and purity.[25],[26] Among the ten herbs of Hridyadashemani, standard monographs are not available for Amra, Lakucha, and Kuvala.
Amra (M. indica L.): The fruits of Amra were drupes, ovoid, green when unripe enclosing a single seed with a stony endocarp. These findings are similar to previous works.[27],[28] Microscopically, the fruit powder showed starch grains, pieces of mesocarp, fibers which are similar to previous reported works.[29] The physicochemical constants of the fruit revealed higher water extractive content than alcoholic, similar to earlier findings.[29] HPLC analysis, revealed that Amra fruits contain gallic acid (15.47 mg/g of extract) and quercetin (1.01 mg/g of extract). Earlier studies have shown high concentrations of polyphenols, including gallic acid and quercetin in the fruits of M. indica.[30],[31]
Amrataka (S. pinnata (L.f.) Kurz)—Macroscopically, the fruits are drupes, containing 1–3 seeds, ovoid, glossy, and green, which was observed in the study.[32] In this study, the powder of Amrataka fruits revealed specific characters that were similar to the observations made in a previous study.[33] The monograph also reveals that the presence of wavy, thick-walled cells of epicarp, different sizes of fibers, annular and spiral vessels, and starch grains.[34] Hence, the observations of the study correlate with previous studies and the pharmacopeial diagnostic characters of the fruit powder.[32],[33],[34] HPLC analysis revealed that Amrataka fruits contain gallic acid (0.75 mg/g of extract) and quercetin (0.08 mg/g of extract). Earlier studies have shown the presence of polyphenols, especially gallic acid, ellagic acid, p-coumaric acid, quercetin, among others.[35]
Lakucha (A. lacucha Roxb. Ex Buch.-Ham)—Fruits of Lakucha are irregularly lobed sorosis with numerous seeds embedded.[36] Microscopically, the fruit powder revealed trichomes, fragments of mesocarp, and fibers. These observations of the fruit relate to previous findings.[37] In this study, physicochemical parameters of the fruit match to previous published literature.[37] HPLC analysis revealed that Lakucha fruits contain gallic acid (3.60 mg/g of extract) and quercetin (0.02 mg/g of extract). Earlier studies have shown the presence of flavonoids, tannins, saponins, steroids, glycosides, triterpenoids, protein, phenolic compounds, and resin.[38]
Karamarda (Carissa carandus L.)—Macroscopically, fruits were berry with white milky latex and 4–6 seeds, which are similar to previous findings.[39],[40] In this study, the powder of Karamarda fruits revealed characters that corroborate with the earlier study.[41] The findings are in line with the diagnostic features of the powder as described in standard monograph.[40] In this study, the alcoholic extractive value (33.33 ± 3.21) is lesser than the pharmacopeial standard (not <35%), and this could be a chance occurrence as the values of three samples (n = 3) were 31%, 32%, and 37%, respectively. HPLC analysis revealed that Karamarda fruits contain gallic acid (0.37 mg/g of extract) and quercetin (0.14 mg/g of extract). Earlier studies have shown the presence of flavonoids and phenolic compounds in fruit juice.[42]
Vrukshamla (Garcinia indica (Thouars) Choisy)—Macroscopically, the fruits were fleshy, juicy berries with seeds covered by aril that are similar to previous reports.[15],[43] The findings of this study revealed diagnostic microscopic features that corroborate with standard monograph.[15],[44] The physicochemical standards of the fruit are within the stated pharmacopeial limits.[15] HPLC analysis revealed that Vrukshamla fruits contain gallic acid (0.50 mg/g of extract) and quercetin (0.24 mg/g of extract). Earlier preliminary phytochemical evaluation has shown the presence of flavonoids, glycosides, saponins, and organic acids in fruit juice.[44]
Amlavetasa (G. pedunculata Roxb.ex Buch.-Ham)—Amlavetasa fruits are fleshy drupes, and the powder microscopy of Amlavetasa fruit is similar to a previous study that reported starch grains, fragments of spiral vessels, fragments of sclereids, and fragments of mesocarp cells.[45] The physicochemical parameters of the fruits are within the limits of pharmacopeial standards[24] and published literature.[45] HPLC analysis revealed that Amlavetasa fruits contain gallic acid (0.11 mg/g of extract) and quercetin (0.01 mg/g of extract). Earlier phytochemical evaluation has shown the presence of flavonoids, polyphenols, and anthocyanins in fruits.[46]
Kuvala(Z. jujuba Mill.)—Macroscopically, Kuvala fruits are ovoid berries, with a rugged ovoid endocarp enclosing seed. In the present powder microscopic study, xylem fibers, epicarp in surface view, and fragments of mesocarp were observed. The physicochemical standards have been recorded in this study and may serve as a standard for future references. HPLC analysis revealed that Kuvala fruits contain gallic acid (0.27 mg/g of extract) and quercetin (0.02 mg/g of extract). Various Zizyphus species are reported to possess various phytochemicals as alkaloids, glycosides, tannins, among others.[47]
Badara (Z. jujuba Lam.)—Macroscopically, fruits were berries with pinkish pulp and a globose endocarp enclosing seeds. These findings of this study are similar to previous reports.[48] The findings of this study reveal diagnostic microscopic features of the fruits that corroborate with standard monograph.[48] In this study, the alcoholic extractive value (28.67 ± 1.53) is lesser than the pharmacopeial standard (not <29%), and the difference is very narrow and could be a chance occurrence as the values of three samples (n = 3) were 27%, 30%, and 29%, respectively. In this study, HPLC analysis revealed that Badara fruits contain gallic acid (0.19 mg/g of extract) and quercetin (0.05 mg/g of extract). Previous studies have reported potential phytochemicals like flavonoids, alkaloids, glycosides, among others.[49],[50]
Dadima (P. granatum L.)—Macroscopically, the seeds of Dadima were ovoid with a juicy testa. The microscopic findings of this study corroborate with previous findings.[51] The physicochemical standards of the fruits fall within the pharmacopeial limits.[52] In this study, HPLC analysis revealed that Dadima fruits contain gallic acid (3.63 mg/g of extract) and quercetin (0.47 mg/g of extract). Previous published works reveal potential phytochemicals from this fruit.[53],[54]
Matulunga (C. medica L.)—Macroscopically the fruits were hesperidium, oblong with a thick rind. The findings of the microscopy are similar to an earlier study, which reported epidermis, fragments of mesocarp and endocarp, in the fruit powder.[55] Physicochemical study revealed that the parameters are within the pharmacopeial limits.[13] In this study, HPLC analysis revealed that Matulunga fruits contain gallic acid (5.61 mg/g of extract) and quercetin (0.01 mg/g of extract). Previous studies reveal the presence of various potential phytochemicals such as flavonoids, phenolic compounds, alkaloids, and others.[55]
These ten herbs are considered as Hridya, meaning it is conducive to the heart. Hence, apart from the preliminary phytochemical evaluation, an attempt was made to evaluate the concentration of specific phytochemicals, like quercetin and gallic acid, which have a role in cardiovascular diseases. Quercetin (a flavonol) is an abundantly available bioflavonoid from plant source that have been proven to possess biological activities like antioxidative, anticarcinogenic, among others.[56] Recent researches have focused their attention on the potential of quercetin in the prevention and treatment of cardiovascular diseases. Studies have shown that quercetin exerts a protective role on the endothelium in atherosclerosis. It has also shown lipid-lowering activity and ability to alter gut microbioata.[57] Various in vitro, in vivo, and clinical studies have shown that quercetin due to its antihypertensive, antiatherosclerotic, antiplatelet activity, and lipid-lowering effects has positive effects on cardiovascular diseases.[58] Previous animal studies reveal that gallic acid improves cardiac dysfunction and fibrosis in pressure overload-induced heart failure in mice.[59] Present HPLC analysis reveals that herbs of Hridyadashemani have varying amounts of quercetin and gallic acid. Among the investigated herbs in the study, quercetin was found in maximum concentration in M. indica extracts (10.06 µg/mL) and least in C. medica L. (1.93 µg/mL). Gallic acid was found in maximum concentration in M. indica extracts (1054.69 µg/mL) and least in G. pedunculata (1.11 µg/mL). Further in vitro, in vivo, and clinical studies of these herbs will reveal its potential in cardiovascular diseases.
| Conclusion | | |
The analytical findings of Amra (M. indica L.), Lakucha (A. lacucha Roxb. Ex Buch.-Ham), and Kuvala (Z. jujuba Mill.) fruits obtained from this study may serve as a standard for future studies. The HPLC finger-print profile of Hridyadashemani with respect to quercetin and gallic acid provides a lead to research in cardiovascular diseases.
Hence, the present preliminary pharmacognostic findings including HPLC finger-print profile would serve to identify, analyze, rationalize, and standardize Hridyadashemani and pave for future evidence-based research in cardiac disorders.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
| Supplementary file | | |
| Quantification of gallic acid and quercetin in given samples by hplc | | |
Test sample:
- Sample 1––Mangifera indica
- Sample 2––Spondias pinnata
- Sample 3––Artocarpus lakoocha
- Sample 4––Carissa carandus
- Sample 5––Garcinia indica
- Sample 6––Garcinia pedunculata
- Sample 7––Ziziphus sativa
- Sample 8––Ziziphus jujube
- Sample 9––Punica granatum
- Sample 10––Citrus medica
Standard: Gallic acid and Quercetin (100 µg/mL each was prepared from the stock of 1 mg/mL by dissolving in methanol)
| HPLC analysis was carried as per the method summarized below: | | |
- I. Instrument: Shimadzhu LC––Prominence 20AT
- II. Column: C18 column 250 mm x 4.6 mm, 5 µm particle
- III. Mobile phase: Linear
- A: HPLC grade ACN (60%)
B: HPLC water (40%)
- IV. Flow rate: 1 mL/min
- V. Injection volume: 10 µL
- VI. Absorbance: 275 nm
Gallic acid and Quercetin was quantified using the peak area after HPLC analysis.
 | Figure S5: HPLC chromatogram of sample 3––Artocarpus lakoocha (10 mg/mL)
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 | Figure S8: HPLC chromatogram of sample 6––Garcinia pedunculata (10 mg/mL)
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[Figure 1], [Figure 2], [Figure F3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9]
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