|Year : 2018 | Volume
| Issue : 4 | Page : 200-207
Anti-inflammatory profile of different plant parts of Agnimantha: A comparative evaluation of two entities enumerated in ayurvedic literature
Narasimha Baba Brindavanam1, Gaya Prasad Kimothi2, Pallu Reddanna2, Rajaram Azad2
1 Dabur Research and Development Centre, Bio Resources Development Group, Ghaziabad, Uttar Pradesh, India
2 Department of Animal Biology, School of Life Sciences, University of Hyderabad, Hyderabad, Telangana, India
|Date of Submission||09-May-2019|
|Date of Decision||03-Dec-2019|
|Date of Acceptance||26-Oct-2021|
|Date of Web Publication||04-Jan-2022|
Dr. Narasimha Baba Brindavanam
Dabur Research and Development Centre, Bio Resources Development Group, 22, Site-IV, Sahibabad, Ghaziabad - 201 010, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Background: Agnimantha is a constituent of the bṛhatpañcamūla (the roots drugs of 5 tree species) which in turn is a part of daśamūla used in Ayurvedic pharmaceutical practices. Traditionally, the concept of bṛhatpañcamūla envisages the usage of root/ root bark of these tree species. By and large, use of stem bark came into vogue many decades ago for this sub-group of daśamūla. Going by descriptions in Ayurvedic lexicon of medicinal plants- two species are considered as Agnimantha viz. Clerodendrum phlomidis L.f. (Fam.: Lamiaceae) and Premna integrifolia L. (Syn. Premna serratifolia L) (Fam.: Lamiaceae). Objective: With an objective to address sustainability concerns associated with use of root or stem bark a comprehensive study was carried out on bṛhatpañcamūla group. This study kept the anti-inflammatory profile of candidate extracts in the centre-stage. As a part of this study, comparative assessment of two species used as Agnimantha was also carried out. Study Methodology: Different plant parts (Root bark, Stem bark, Leaves and Young roots) of C. phlomidis and P. integrifolia were collected from different parts of India. Each sample was extracted successively into four solvents. These extracts were evaluated for their anti-inflammatory profile using a battery of in-vitro assays, involving inhibition of regulatory enzymes like 5-lipoxygenase (5-LOX), cyclooxygenase 1&2 (COX-1 & COX-2) and analysis of the expression of pro- and anti-inflammatory cytokines in LPS-stimulated RAW 264.7 cells.Result and Conclusions: Both the species were observed to exhibit anti-inflammatory activity of varied degrees in this study. However, the sample of 12 months old roots of P. integrifolia was found to possess profound effect on all markers of inflammation. This sample was followed by 36 months old roots of C. phlomidis in terms of anti-inflammatory profile. Basing on these observations, the study suggests the use of 12 months roots of P. integrifolia (often referred to as Bṛhat-agnimantha) as part of daśamūla. Since the harvesting cycle is of 12 months, it is possible to produce the roots using High-Density Short-Term plantation protocols to address the sustainability concerns associated with use of root or stem bark.
Keywords: 5-Lipoxygenase, agnimantha, anti-inflammatory bṛhatpañcamūla, Clerodendrum, cyclooxygenase 1, 2, cytokines, daśamūla, Premna
|How to cite this article:|
Brindavanam NB, Kimothi GP, Reddanna P, Azad R. Anti-inflammatory profile of different plant parts of Agnimantha: A comparative evaluation of two entities enumerated in ayurvedic literature. Ancient Sci Life 2018;37:200-7
|How to cite this URL:|
Brindavanam NB, Kimothi GP, Reddanna P, Azad R. Anti-inflammatory profile of different plant parts of Agnimantha: A comparative evaluation of two entities enumerated in ayurvedic literature. Ancient Sci Life [serial online] 2018 [cited 2022 Jul 3];37:200-7. Available from: https://www.ancientscienceoflife.org/text.asp?2018/37/4/200/334723
| Introduction|| |
Daśamūla is an important fixed dose combination enumerated in pharmaceutical practices of Ayurveda. Described in suśrutasaṃhitā for the first time, the group comprises 10 root drugs which are further divided into two subgroups, viz., Bṛhatpañcamūla (root drugs of 5 tree species) and Laghupañcamūla (root drugs of 5 herbaceous species). As a concept, Daśamūla envisages the use of roots for both these subgroups. However, in subsequent interpretations of terminology, it was explicitly suggested to use root bark in case of the former subgroup. As a result, Bṛhatpañcamūla practically refers to roots barks of five tree species, viz., Bilva, Agnimantha, syonāka, Pāţala, and Gambhārī. While the root bark or roots is authentic plant part for these species, trade practices largely involve the supply of stem bark during last few decades. The first sub group – the Bṛhatpañcamūla – is associated with two broad complexities, viz., sustainability concerns and nomenclature.
From utilization purpose, Daśamūla as a group is widely used in a number of classical ayurvedic formulations. As a result, the estimated consumption volumes for each of the member species range between 2000 and 2500 MT per annum. Given this kind of volume demands, the use of roots/root bark or stem bark in case of Bṛhatpañcamūla might not be sustainable more so, when the species is endemic in its occurrence. There is a pressing need to address such usage practice.
Nomenclature of Daśamūla members
Second, daśamūla as group is also riddled with technical issues on account of botanical nomenclature for some of its member species. Ayurvedic Nighantus enumerated more than one species for Agnimantha, syonāka, and pṛśniparṇī of which botanical entities of Agnimantha are the subject matters of this study.
While describing the local applications for treatment of ūrustambha, Charaka mentions two entities, tarkārī and Agnimantha distinctly. On basis this description, scholars opined that Agnimantha relates to Premna species while tarkārī refers to Clerodendrum species. Daśamūla group described by suśruta included the name of Agnimantha. On this basis, Premna species need to be considered as part of this combination and the other entity - tarkārī (viz., Clerodendrum sp.) has no role in it. rājanighaṇṭu described two types of Agnimantha - kṣudra (or laghu) and Bṛhat. It may be stated that different authorities in modern times assigned different botanical species for Agnimantha. Nadkarni correlated it to Clerodendrum inerme (which is extensively grown as ornamental hedges) while Sivarajan suggested it, to be Premna corymbosa. Ayurvedic Pharmacopoeia of India recognized Clerodendrum phlomidis as Agnimantha and included a pharmacopoeia monograph. Contrary to this status, Ayurvedic Formulary of India - which is also a regulatory document for the purpose of classical Ayurvedic formulations - recognizes Premna integrifolia as Agnimantha while C. phlomidis and Premna mucronata are suggested to be its botanical substitutes.
Biological activities of Agnimantha
Both the botanical entities of Agnimantha are reported to exhibit anti-inflammatory activities. Root bark and leaves of C. phlomidis were evaluated for anti-inflammatory and anti-arthritic effects using in vivo experimental models.,, In vivo experimental models were used to demonstrate anti-inflammatory and anti-arthritic activities of wood, roots, and flowers of P. integrifolia.,, Analgesic activity of its leaves is also reported in another study. Both entities of Agnimantha were reported to exhibit hepatoprotective activity., Further, both the species are evaluated for their effects on metabolic conditions as well in different studies. Both the species are reported to exhibit antiobesity activity in separate studies., In addition, C. phlomidis was reported for its effect in diabetes, while P. integrifolia is reported to be effective against hyperlipidemia, and also as a cardioprotective agent.
To address the issues in resource management of Bṛhatpañcamūla, there is a pertinent need to identify alternative plant parts. Concurrently, it is also desirable to identify preferable plant species for Agnimantha. In this direction it is necessary that, the conceptual approach must be centered around biological activity of Agnimantha. Hence, the objectives of this study are defined as under:
- To evaluate the anti-inflammatory profile of different plant parts of Agnimantha
- To compare (in terms of anti-inflammatory profile) two botanical entities, viz., C. phlomidis L.f. (Syn. C. multiflorum G.don) (Laghu-Agnimantha [LA]) and P. integrifolia L. (Syn. P. serratifolia L.) Bṛhat-Agnimantha [BA]) both belonging to family Lamiaceae (Verbenaceae in earlier times).
| Materials and Methods|| |
Collection of study samples and extraction
The study samples were collected by the scientists from one of the study partners, Dabur Research and Development Centre (DRDC).
Sampling of Laghu-Agnimantha
Root bark sample was collected from Madhya Pradesh (MP) and stem bark was collected from both MP and Gujarat. However, collecting samples of bark care was taken to minimize damage to the plant. Two types of sources were approached for purpose of collecting young roots (YRs) of four predefined ages (1, 1.5, 2, and 3 years). The first source comprised newly commissioned project sites of resource augmentation in the states of Gujarat (GJ2 and GJ3), Maharashtra (MH), and Odisha (OR1 and OR2). The second source of sampling is comprised of a limited plantation activity, speciallly for the purpose of sampling required for the study. GJ1 and KA samples represented this group.
Sampling of Bṛhat-Agnimantha
Sample of mature roots was obtained from Gujarat. Samples of YR of predefined age (1, 1.5, 2, and 3 years) were obtained through a limited plantation activity initiated, speciallly for the purpose of study in the states of Gujarat, Karnataka, and Maharashtra.
Botanical identity of the samples was authenticated by Dr. S. K. Srivastava, Scientist-E, BSI, Dehradun. All the reference samples are being maintained in the Pharmacognosy Laboratory of DRDC. The herbarium sheet of voucher specimen was deposited with Northern Regional Centre, Botanical Survey of India, Dehradun (Accession nos. 2315716 for LA and 2315717 for BA) [Table 1 of supplementary information for precise locations of sampling].
Each sample was extracted into four solvents, viz., petroleum ether (PE), ethyl acetate (EA), ethanol (ET), and aqueous (AQ) using a sequential extraction method [Table 2 of supplementary information for extractive values of individual samples in different solvents]. AR grade solvents supplied by standard manufacturers were used for extraction purposes. The biological activity of all extracts was assessed in the Department of Animal Biology, School of Life Sciences, University of Hyderabad.
The extracts were screened for anti-inflammatory profile in a two-layered screening method. All the extracts (120 of LA and 52 extracts of BA) were screened for their effect on cyclooxygenases (COX-1 and 2) and 5-lypoxygenase (5-LOX) using noncellular enzymatic test model. The MIC was calculated. Further, all these extracts were assayed lymphocyte proliferation activity (LPA) and methyl thiazolyl tetrazolium (MTT) cytotoxicity.
During the second phase, the selected extracts were tested for their inhibitory effect on three of pro-inflammatory cytokines (interleukin-1 beta [IL-1b], IL-6, and MIP-1a) and one of the anti-inflammatory cytokine, IL-2. These tests were performed in vitro, using RAW-264.7 cell line (mouse macrophages).
Reagents and chemicals
Culture media, antibiotics (penicillin/streptomycin), and concavalin-A (Con-A) were purchased from HiMedia Laboratories (Mumbai, India), fetal bovine serum (FBS) from Hyclone. Ficoll Histopaque, lipopolysaccharide (LPS), and MTT were purchased from Sigma-Aldrich (St Louis, MO 63103, USA). The TMPD (N, N, N', N'-tetramethyl p-phenylenediamine), hematin, and Tween 20 were purchased from Sigma-Aldrich and arachidonic acid was purchased from Nu-check Prep, Inc. (MN, USA). The dimethyl sulfoxide (DMSO) used was of HPLC grade. All the solutions were prepared in de-ionized distilled water. All other reagents used in the studies were of standard quality.
Extraction and isolation of cyclooxygenases
COX-1 was isolated from ram seminal vesicles and preparation of microsomes was carried out as per method described by Hemler and Lands. Spodoptera frugiperda-9 cells infected by recombinant Baculovirus were used for expression of human recombinant COX-2 using a slightly modified method reported earlier. An outline of these procedures is also provided in our earlier research communication. Chromogenic assay procedure described by Copeland et al. was used to measure the enzymatic activities of both COX-1 and COX-2. This procedure is based on the oxidation of TMPD during the reduction of PGG2 to PGH2., The assay mixture comprising 100 mM Tris–HCl buffer (pH 8.0), 5 mM, hematin, 5 mM EDTA, enzyme (COX-1 or COX-2), and the test extract was preincubated at 25°C for 5 min. The reaction was initiated by the addition of substrate, arachidonic acid, and TMPD. Total volume of the reaction mixture was made up to 1 ml. The enzyme activity was determined by estimating the rate of TMPD oxidation for the first 60 s of the reaction by following the increase in absorbance at 610 nm. A low rate of nonenzymatic oxidation, observed in the absence of COX-1 and COX-2, was subtracted from the experimental value while calculating the percent inhibition.
Purification and assay of 5-lipoxygenase
5-LOX was isolated from potato tubers and was purified - as per the procedure described by Reddanna et al. Polarographic method with a Clark's oxygen electrode on Strathkelvin Instruments (Model 782, RC-300) was used for measurement of enzyme activity. The reaction mixture contained 50–100 μl of enzyme and 10 μl of 40 mM substrate (arachidonic acid). The final volume was made to 3 ml with 100 mM phosphate buffer (pH 6.3). Since the lipoxygenases are oxygen-consuming enzymes, the rate of decrease in oxygen was taken as a measure of enzyme activity. Assay was standardized using nordihydroguaiaretic acid (NDGA) which is a selective LOX inhibitor. Reaction was carried out at 25°C, and the maximum slope generated was taken for calculating enzyme activity. The activity was expressed as units/mg protein, where one unit is defined as one micromole of oxygen consumed per minute.
Cell culture and treatment
RAW 264.7 cell line was obtained from National Centre for Cell Science, Pune, India. Cell culture was done in RPMI 1640 media supplied with 10% heat-inactivated FBS and 100 U/ml penicillin and 100 μg/μl streptomycin. The culture was maintained at 37°C in a humidified atmosphere with 5% CO2. Cells were propagated by splitting and changing the media twice a week.
Methyl thiazolyl tetrazolium assay
MTT assay performed by the method described by Mossman. Effects of extracts or LPS or standards was assessed using LPS-challenged cells and untreated cells as control. For this purpose, 5 × 103 RAW 264.7 cells were grown in 96-well plates for 24 h and subsequently were pretreated with plant extracts at three different concentrations (100, 10 and 1 μg/mL) or standards (celecoxib, indomethacin, and NDGA) for 3 h and then challenged with LPS at 1 μg/mL for 48 h. Untreated cells have served as control for LPS. After 48 h incubation, the cell supernatant was carefully aspirated and 20 μL of MTT (5 mg/ml in PBS) was added in each well and was incubated for an additional 3 h at 37°C. Tetrazolium crystals were solubilized by adding 50 μL of DMSO to each well and agitated for 5 min on a rocker. Absorbance was read at 570 nm in a multimode reader (SynergyMx, BioTek). Percent growth of cells was calculated using control as reference.
Lymphocyte proliferation assay
A sample of blood was collected afresh from a healthy human volunteer after obtaining IEC permission (IEC/2017/44) and a written consent by the donor. Lymphocytes were isolated from fresh blood using Ficoll–Histopaque (Sigma) method. Lymphocytes were washed with PBS and re-suspended in complete media DMEM along with addition of 50 μM β-mercaptoethanol. Cells were seeded at the density of 5 × 103 cells in each well in 96-well plates and grown for 16 h at 37°C and 5% CO2. Cells were treated with the plant extracts of three different concentrations (100, 10, and 1 μg/mL) along with the standards viz., 25 μM celecoxib, 10 μM indomethacin, and 10 μM NDGA in duplicates and incubated for 24 h. Cells treated with Con-A (4 μg/mL) served as positive control and without Con-A served as control. After 24 h, 20 μL of MTT (5 mg/mL in PBS) was added in each well and incubated for an additional 3 h at 37°C. After incubation, 50 μL of DMSO was added in each well to dissolve the formazan crystals. Absorbance was read at 570 nm in multimode reader (SynergyMx, BioTek). The percent growth of lymphocytes was calculated using control as reference.
Analysis of pro- and anti-inflammatory cytokines
Effect of selected extracts on expression of pro- and anti-inflammatory markers IL-1β, IL-2, IL-6, and MIP1-α was evaluated using LPS-stimulated RAW 264.7 cells. For this purpose, 2 × 105 cells per well was seeded in six-well plates and was allowed to grow for 16h. They were then incubated with plant extracts at their IC50 concentration (from COX-2 enzyme assay studies) for 1 h. The cells were then activated with LPS (1 μg/mL) for 5 h and the supernatants were collected for further cytokine analysis. Cytokine expression was measured by ELISA using commercially available kits (R and D Systems, MN, USA). Prior to measures with the extracts, method was standardized using Celecoxib. At least, a 50% inhibition in relation to LPS-treated cell is considered as effective for pro-inflammatory cytokines (IL-1α, Il-6 and MIP-1α). At least, a twofold increase is considered as effective in case of IL-2 induction.
Data were expressed as mean ± standard deviation of three individual experiments, and a P ≤ 0.05 was considered as statistically significant.
| Results|| |
Effects of various extracts of Laghu and Bṛhat-Agnimantha on the inhibition of cyclooxygenases and/or 5-lipoxygenase, lymphocyte proliferation activity assay and methyl thiazolyl tetrazolium in primary screens
A total of 30 samples were collected for LA, while 13 samples were collected in case of BA from different locations of sampling. Thus, a total of 120 extracts of LA and 52 extracts of BA were taken up for initial screening for inhibitory effects on COX and 5-lipoxygenase. Concurrently, each of the extract was also evaluated for cytotoxic (MTT assay) and mitogenic (LPS assay) properties. The outcomes of these screening tests are shown in [Figure 1]. It may be observed from the figure that none of the LA extracts had any effect on 5-LOX while six extracts of BA could inhibit the pro-inflammatory enzyme. On the other hand, 17 extracts of LA were observed to inhibit COX-2 in a selective manner – whereas, none of the BA extracts exhibited such selectivity (Refer to Supplementary Information for MIC values in the Master Data).
|Figure 1: Effects of various extracts of LA and BA on the inhibition of cyclooxygenases and/or 5-lipoxygenase in primary screens. LA: Laghu Agnimantha, BA: Bṛhat-agnimantha, COX-1: Cyclooxygenase 1, 5-LOX: 5-lypoxygenase|
Click here to view
Effect of selected extracts of Laghu-Agnimantha and Bṛhat-Agnimantha on Expression of pro- and anti-inflammatory cytokines (interleukin-1 beta, interleukin-6, MIP-1α, and interleukin-2) in RAW-264.7 cell line challenged with lipopolysaccharide
A total of 36 extracts of LA and 17 of BA were taken up for assessment of their effect on cytokine expression. At least, a 50% reduction in pro-inflammaotry cytokines under test conditions is considered as “inhibitory effect”. Similarly, a two-fold increase is considered to qualify for Induction effect on anti-inflammatory cytokine, IL-2. 22 of 36 extracts of LA were found to influence the expression of at least one cytokine in case of [Figure 2]. 9 out of 17 extracts of BA were observed to influence expression of at least, one cytokine in LPS-challenged macrophages in [Figure 3].
|Figure 2: Effects of Laghu Agnimantha on cytokine expression in RAW 264.7 cell line challenged with LPS. Code for sampling sources: L: Laghu, GJ1, 2, 3: Gujarat sampling sites 1, 2 or 3, KA: Karnataka, MP: Madhya Pradesh, OR1, OR2: Orissa sampling sites 1 or 2. Codes of plant parts: RB: Root bark, SB: Stem bark, LF: Leaves, YR-1, YR-1.5, YR-2, and YR-3: Young roots 12 months, Young roots 18 months, Young roots 24 months, and young roots 36 months age, respectively. Codes for extracts: 1: Petroleum benzene extract, 2: Ethyl acetate extract, 3: Ethanolic extract, 4: Aqueous extract. For example: L/GJ3/YR-3/2 denotes the ethyl acetate extract of 36-month-old Young roots of Laghu Agnimantha drawn from 3rd sampling site of Gujarat. Qualification Criteria: A minimum 50% inhibition in pro-inflammatory cytokines (IL-1β, IL-6, and MIP-1α) or two-fold increase in expression of IL-2 (which is anti-inflammatory cytokine)|
Click here to view
|Figure 3: Effects of extracts of BA on cytokine expression in RAW 264.7 cell line challenged with LPS. Code for sampling sources: IL-1βIL-1ing source BA: Bṛhat-Agnimantha, GJ 1: Gujarat sampling site 1, KA: Karnataka, MH: Maharashtra. Codes of Plant Parts: RB: Root bark, SB: Stem bark, LF: Leaves, YR-1, YR-1.5, YR-2 and YR-3: Young roots 12 months, young roots 18 months, young roots 24 months and young roots 36 months age respectively. Codes for extracts: 1: Petroleum benzene extract, 2: Ethyl acetate extract, 3: Ethanolic extract, 4: Aqueous extract. For example: B/KA/YR-1/2 denotes the ethyl acetate extract of 12-month-old Young roots of Bṛhat-Agnimantha drawn from Karnataka. Qualification criteria: A minimum 50% inhibition in pro-inflammatory cytokines (IL-1β, IL-6, and MIP-1α) or two-fold increase in expression of IL-2 (which is anti-inflammatory cytokine)|
Click here to view
It may be observed from [Figure 2] and [Figure 3] that the effect of extracts of LA and BA on cytokine expression is not consistent. In case of LA, the effect was prominent on anti-inflammatory cytokine IL-2 induction with 21 extracts promoting its expression. In case of BA, the inhibitory effect on IL-6 has been more prominent with 9 extracts influencing it in a desired fashion.
Only three extracts of LA and one extracts of BA exhibited desirable effects on the expression of all four cytokines in a consistent manner. Hence, it is necessary to take a closer look on these four extracts. [Figure 4], [Figure 5], [Figure 6], [Figure 7] show the effect of these selected extracts representing both LA and BA on four of the tested cytokines.
|Figure 4: Effect of selected extracts of LA and BA on expression of pro-inflammatory cytokine (IL-1β) in RAW-264.7 Cell line challenged with LPS. LPS: Lipopolysaccharide, IL-1βInterleukin-1 beta LA: Laghu Agnimantha, BA: bṛhat Agnimantha, GJ3: Gujarat Sampling Site-3, GJ2: Gujarat sampling site-2, KA: Karnataka, YR: Young roots, YR-1: One year of YR, YR-3: Three years of YR, 1: Petroleum ether extract, 2: Ethyl acetate extract. #P < 0.001 for comparison between LPS alone versus un-treated, *P < 0.001 compared between cells treated with LPS in the presence of extract/celecoxib versus LPS alone|
Click here to view
|Figure 5: Effect of selected extracts of LA and BA on expression of pro-inflammatory cytokine (IL-6) IL-6pression of pro-inflammatory cytokine h #P < 0.001 for comparison between LPS alone versus un-treated. *P < 0.001 compared between cells treated with LPS in the presence of extract/celecoxib versus LPS alone. LA: Laghu Agnimantha, B: bṛhat Agnimantha, LPS: Lipopolysaccharide, IL-1βLipopolysaccha GJ3: Gujarat sampling site-3, GJ2: Gujarat sampling site-2, KA: Karnataka, YR: Young roots, YR-1: One year of YR, YR-3: Three years of YR, 1: Petroleum ether extract, 2: Ethyl acetate extract|
Click here to view
|Figure 6: Effect of selected extracts of LA and BA on expression of pro-inflammatory cytokine (MIP-1α) in RAW-264.7 Cell line challenged with LPS. #P < 0.001 for comparison between LPS alone versus un-treated. *P < 0.001 compared between cells treated with LPS in the presence of extract/celecoxib versus LPS alone. LPS: Lipopolysaccharide, LA: Laghu Agnimantha, BA: bṛhat Agnimantha, GJ3: Gujarat sampling site-3, GJ2: Gujarat sampling site-2, KA: Karnataka, YR: Young Roots, YR-1: One year of YR, YR-3: Three years of YR, 1: Petroleum ether extract, 2: Ethyl acetate extract|
Click here to view
|Figure 7: Effect of selected extracts of LA and BA on expression of anti-inflammatory cytokine (IL-2) IL-2ssion of acell line challenged with LPS. *P < 0.001 compared between cells treated with LPS in the presence of extract/celecoxib versus un-treated cells. LPS: Lipopolysaccharide, LA: Laghu Agnimantha, BA: bṛhat Agnimantha, GJ3: Gujarat sampling site-3, GJ2: Gujarat sampling site-2, KA: Karnataka, YR: Young roots, YR-1: One year of YR, YR-3: Three years of YR, 1: Petroleum ether extract, 2: Ethyl acetate extract|
Click here to view
It may be observed that LPS treatment causes significant increase of three pro-inflammatory cytokines in RAW-264.7 cell line. The extracts of LA and BA significantly reduced such expression. It may further be noted that, there is no significant difference between the test samples in terms of their inhibitory effect on cytokine expression by RAW-264.7 cell line. Similarly, LPS-induced decrease in IL-2 expression is effectively countered by the test extracts. However, there is no visible difference of such desirable effect between the four extracts.
| Discussion|| |
This study was a part of a major network investigation to address the sustainability concerns associated with the use of root bark and/or stem bark in case of bṛhatpañcamūla. In fact, the group name bṛhatpañcamūla (root drugs of 5 tree species) correlates to the use of “roots from prescribed trees/shrubs.” However, the interpretations to terminology during later centuries sought the use of root bark when the roots of tree/shrubs are suggested in the text. Thus, the name bṛhatpañcamūla implies the use of “root barks” of these species by default. Obviously, there is technical requirement of using root bark in case of the subject species of Agnimantha too, but the practice is unsustainable on long run. Hence, there is a visible need to identify sustainable plant parts for Agnimantha in general for replacement of root bark. The concept of Abhāvapratinidhi dravya is well evolved – wherein, alternative species having similar properties can be taken up for use – in case, the recommended original plant drug is not available. By etymology, Abhāva refers to shortage (of recommended drug) and pratinidhi refers to substitution. This concept primarily touched upon the selection of alternate plant species, but also provided insights into the use of alternative plant parts as Abhāvapratinidhi.
The present study relating to Agnimantha represents a well-evolved concept of plant part substitution laid down in Ayurveda. The concept is structured in the foundations of biological activities. While working upon identification of sustainable plant parts, it is also felt necessary to examine both the botanical entities of Agnimantha – to take holistic view on the subject of investigation. Therefore, the results of the study maybe analyzed and discussed from two distinct perspectives.
Choice between Laghu and Bṛhat Agnimantha
As mentioned elsewhere in this paper, two botanical entities are enumerated as Agnimantha in different literary sources. A comparison of biological activities of both entities is desirable when the objective is to examine the resource from a sustainability perspective. However, the purpose of this comparison is not to resolve as to which species actually constitutes the entity, Agnimantha. Our study observed that both the C. phlomidis and P, integrifolia possess anti-inflammatory activity. Four extracts of C. phlomidis and one extract of P. integrifolia were noted to exhibit a comprehensive anti-inflammatory profile. These extracts were observed to inhibit enzymatic mediators of inflammation. Further, they were noted to modulate cytokine expression in mouse macrophages (RAW-264.7 cell line) challenged by LPS.
Another comprehensive study concerning Agnimantha was submitted to Gujarat University. In vivo experimental models were used to compare anti-inflammatory activity of C. phlomidis and P. integrifolia. A part of this study also reported the immunomodulatory profile of the subject species and isolation of clerodendrin-A from the roots of both the species. These reported studies support our observations. However, we observed that the biological activity was optimized in the roots of 3 years (YR-3) in case of LA. In case of BA, the activity was noted to be optimal in 1-year old roots (YR-1).
Plant part substitution
A battery of anti-inflammatory assays was used to screen different plant parts. This battery of tests was designed to elicit the anti-inflammatory profile of the study candidates in a comprehensive manner. Basing on the outcomes, the young roots (YR-1 of P. integrifolia or YR-3 of C. phlomidis) may be considered as alternative plant parts. The pharmacognostic features of of these two qualified samples are shown in supplementary [Figure 1] (for LA) and supplementary [Figure 2] (for BA). This outcome provides an opportunity to produce young roots through high-density short-term plantation schemes. A systematic cultivation of species and harvesting the roots at prescribed age of 1 or 3 years (as the case may be) is expected to address the sustainability concerns of harvesting mature roots for root bark or stem bark.
| Conclusion|| |
On the basis of anti-inflammatory profiles elicited in this study, it may state that both entities of Agnimantha used in Ayurveda (Laghu and Bṛhat- C. phlomidis and P. integrifolia, respectively) are comparable in terms of biological activity. This study was focused on evaluation of young roots of different ages for both the species to consider sustainability aspects. From this perspective, the use of YR-1 of BA (P. integrifolia L. [Syn. P. serratifolia L.]) seems to be a logical option. For sustainable supply, these roots can be cultivated under high-density plantation scheme.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Sushrita. Sushrita Samhita, Sutrasthana, 38/33, 34: Hindi Trans. & Comm. by Ambika Datta Sastry. 6th
ed. Varanasi: Chaukhambha Sanskrit Sansthan; 1987. p. 147.
Sharngdhara. Sharngadhara Samhita Prathama Khanda, 1/60, 61, Eng. Trans. & Commentary by Srikanta Murty. 1st
ed. Varanasi: Chaukhambha Orientalia; 1984. p. 9.
Ved DK, Goraya GS. Demand & Supply of Medicinal Plants in India (NMPB & FRLHT). Dehradun: Bishen Singh Mahendrapal Singh; 2008.
Aparna S, Ved DK, Lalitha S, Venkatasubramanian P. Botanical identity of plant sources of Daśamūla drugs through an analysis of published literature. Anc Sci Life 2012;32:3-10.
Agnivesa. Charaka Samhita, Chikitsasthana, 27/54, 55, English Translation and Comm. by PV Sharma. 2nd
ed. Varanasi: Choukhambha Orientalia, Varanasi; 1992. p. 459-60.
Thakur BS, Chunekar KC. In Glossary of Vegetable Drugs in Brihattrayi, Varanasi. 1st
ed. Varanasi: Chaukhambha Sanskrit Series Office; 1972. p. 4-5.
Narahari P. Raja Nighantu, Shalmalyadi Varga, 22/15: With Dravyaguna Prakashika Hindi Commentary by Indradeo Tripathi. 1st
ed. Varanasi: Krishna Das Academy; 1982. p. 268-9.
Nadkarni AK, editor. KM Nadkarni's Indian Materia Medica, Bombay. 2nd
Reprint of 3rd
ed., Vol. 1. Bombay: Bombay Popular Prakashan; 1982. p. 1009.
Sivarajan VV, Balachandran I. Ayurvedic Drugs and their Plant Sources. 1st
ed. New Delhi: Oxford & International Book House Publishing Company; 1994. p. 21.
Anonymous. Ayurvedic Pharmacopoeia of India. Part-I., 1st
ed., Vol. 3. New Delhi: Government of India, Ministry of Health & Family Welfare, Department of AYUSH; 2001. p. 3.
Anonymous. Ayurvedic Formulary of India. Part-I., 2nd
ed. New Delhi: Government of India, Ministry of Health & Family Welfare; 2003. p. 307.
Parekar RR, Dash KK, Marathe PA, Apte AA, Rege NN. Evaluation of anti-inflammatory activity of root bark of Clerodendrum phlomidis
in experimental models of inflammation. Int J Appl Biol Pharm Tech 2012;3:54-60.
Killimozhi D, Parthasarathy V, Amuthavalli N. Effect of Clerodendrum phlomidis
on adjuvant induced arthritis in rats – A radiographic densitometric analysis. Int J Pharm Tech Res 2009;1:1434-41.
Babu NP, Pandikumar P, Ignacimuthu S. Lysosomal membrane stabilization and anti-inflammatory activity of Clerodendrum phlomidis
L.f., a traditional medicinal plant. J Ethnopharmacol 2011;135:779-85.
Rajendran R, Krishnakumar E. Anti-arthritic activity of Premna serratifolia
Linn., wood against adjuvant induced arthritis. Avicenna J Med Biotechnol 2010;2:101-6.
Gokani RH, Lahiri SK, Santani DD, Shah MB. Evaluation of anti-inflammatory and antioxidant activity of Premna integrifolia
root. J Compl Int Med 2011;8:25.
Rajagopal PL, Aneesha S, Sreejith KR, Kiron SS, Premalatha K. Antioxidant and anti inflammatory studies on the flowers of Premna serratifolia
Linn. Int J Adv Pharm Biol Chem 2014;3:679-82.
Karmakar UK, Pramanik S, Sadhu SK, Shill MC, Biswas SK. Assessment of analgesic and antibacterial activity of Premna integrifolia
(Family: Verbenaceae) leaves. Int J Pharm Sci Res 2011;2:1430-5.
Verma A, Ahmed B. Anti-hepatotoxic activity of Clerodendrum phlomidis
. Int J Pharm Tech Res 2009;1:1028-31.
Vadivu R, Jerad SA, Girinath K. Bhoopathi Kannan P, Vimala R, Sathish Kumar NM. Evaluation of hepatoprotective and in-vitro
cytotoxic activity of leaves of Premna serratifolia
Linn. J Sci Res 2009;1:145-52.
Chidrawar VR, Patel KN, Chitme HR, Shiromwar SS. Pre-clinical evolutionary study of Clerodendrum phlomidis
as an anti-obseity agent against high fat induced C57BL/6J mice. Asian Pac J Trop Biomed 2012:S1509-19.
Mali Prashant Y. Effect of aqueous enriched fraction of Premna integrifolia
root against cafeteria diet induced obesity in Swiss albino mice. Int J Green Pharm 2013;7:315-21. [Full text]
Chaturvedi GN, Subramaniyam PR, Tiwari SK, Singh KP. Experimental and clinical studies on diabetes mellitus evaluating the efficacy of an indigenous oral hypoglycaemic drug-Arani (Clerodendron phlomidis
). Anc Sci Life 1984;3:216-24.
Patel MJ, Patel JK. Evaluation of anti-hyperlipidaemic activity of Premna integrifolia
using experimental animal model. Int J Res Phytochem Pharmacol 2011;1:146-9.
Patel MJ, Patel JK. Evaluation of anti-hyperlipidaemic activity of Premna integrifolia
on nicotine induced hyperlipidaemia in rats. Int J Pharm Biosci 2012;3:226-342.
Rajendran R, Basha SN. Cardioprotective effect of ethanol extract of stem bark and stem wood of Premna serratifolia
). Res J Pharm Tech 2008;1:487-91.
Hemler M, Lands WE. Purification of the cyclooxygenase that forms prostaglandins. Demonstration of two forms of iron in the holoenzyme. J Biol Chem 1976;251:5575-9.
Reddy CM, Bhat VB, Kiranmai G, Reddy MN, Reddanna P, Madyastha KM. Selective inhibition of cyclooxygenase-2 by C-phycocyanin, a biliprotein from Spirulina platensis
. Biochem Biophys Res Commun 2000;277:599-603.
Azad R, Vanaja GR, Preeti V, Aparna R, Reddy GV, Anil Kumar K, et al
. Anti-inflammatory profile of Aegle marmelos
(Bilva) with special reference to young roots grown in different parts of India. J Ayurveda Int Med 2018;9:90-8.
Copeland RA, Williams JM, Giannaras J, Nurnberg S, Covington M, Pinto D, et al.
Mechanism of selective inhibition of the inducible isoform of prostaglandin G/H synthase. Proc Natl Acad Sci U S A 1994;91:11202-6.
Egan RW, Paxton J, Kuehl FA Jr. Mechanism for irreversible self-deactivation of prostaglandin synthetase. J Biol Chem 1976;251:7329-35.
Pagels WR, Sachs RJ, Marnett LJ, Dewitt DL, Day JS, Smith WL. Immunochemical evidence for the involvement of prostaglandin H synthase in hydroperoxide-dependent oxidations by ram seminal vesicle microsomes. J Biol Chem 1983;258:6517-23.
Reddanna P, Whelan J, Maddipati KR, Reddy CC. Purification of arachidonate 5-lipoxygenase from potato tubers. Methods Enzymol 1990;187:268-77.
Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55-63.
Sitz KV, Birx DL. Lymphocyte proliferation assay. Methods Mol Med 1999;17:343-53.
Bhavamishra, Bhavaprakasha, Pradhamakhanda, Hindi Translation & Commentary by Mishra B. S. & Vaisya R. L. 5th
ed. Varanasi: Chaukhambha Sanskrit Santha; 1988. p. 181-3.
Yogaratnakara with Vidyotini Hindi Commentary by Sastry L, edited by Sastry B. S. 4th
ed. Varanasi: Chaukhambha Sanskrit Sansthan; 1988. p. 171-5.
Vallabhacharya. Vaidyachintamani with Hindi Translation & Commentary by Sharma R. N. & Sharma S. 2nd
ed., Ch. 25. Delhi: Chaukhambha Sanskrit Pratishtan; 1996. p. 661.
Gokani Rina H. Pharmacognostical, Phytochemical and Pharmacological Studies on Clerodendrum phlomidis
, Premna integrifolia
, Clerodendrum serratum
and Premna herbacea
, Thesis Submitted for Award of Ph. to Faculty of Pharmacy, Gujarat University, Ahmedabad (T3005) Under Guidance of Shah Mamta B; 2007. Available from: http://shodhganga.inflibnet.ac.in/handle/10603/48518
. [Last accessed on 2017 Mar 07].
Gokani RH, Lahiri SK, Santani DD, Shah MB. Evaluation of immunomodulatory activity of Clerodendrum phlomidis
and Premna integrifolia
root. Int J Pharmacol 2007;3:352-6.
Gokani RH, Shah MB. Isolation and estimation of clerodendrin-A in Clerodendrum phlomidis
and Premna integrifolia
root. J Pharm Res 2009;8:9-11.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]