Ancient Science of Life

: 2018  |  Volume : 37  |  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

Correspondence Address:
Dr. Narasimha Baba Brindavanam
Dabur Research and Development Centre, Bio Resources Development Group, 22, Site-IV, Sahibabad, Ghaziabad - 201 010, Uttar Pradesh


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.

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;37:200-207

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 Nov 28 ];37:200-207
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Daśamūla is an important fixed dose combination enumerated in pharmaceutical practices of Ayurveda. Described in suśrutasaṃhitā[1] 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,[2] 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.

Sustainability concerns

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.[3] 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[4] 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.[5] On basis this description, scholars opined that Agnimantha relates to Premna species while tarkārī refers to Clerodendrum species.[6] 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[7] 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[8] correlated it to Clerodendrum inerme (which is extensively grown as ornamental hedges) while Sivarajan[9] suggested it, to be Premna corymbosa. Ayurvedic Pharmacopoeia of India recognized Clerodendrum phlomidis as Agnimantha and included a pharmacopoeia monograph.[10] Contrary to this status, Ayurvedic Formulary of India[11] - 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.[12],[13],[14] In vivo experimental models were used to demonstrate anti-inflammatory and anti-arthritic activities of wood, roots, and flowers of P. integrifolia.[15],[16],[17] Analgesic activity of its leaves is also reported in another study.[18] Both entities of Agnimantha were reported to exhibit hepatoprotective activity.[19],[20] 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.[21],[22] In addition, C. phlomidis was reported for its effect in diabetes,[23] while P. integrifolia is reported to be effective against hyperlipidemia[24],[25] and also as a cardioprotective agent.[26]

Study objectives

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 AgnimanthaTo 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.[27] Spodoptera frugiperda-9 cells infected by recombinant Baculovirus were used for expression of human recombinant COX-2 using a slightly modified method reported earlier.[28] An outline of these procedures is also provided in our earlier research communication.[29] Chromogenic assay procedure described by Copeland et al. was used to measure the enzymatic activities of both COX-1 and COX-2.[30] This procedure is based on the oxidation of TMPD during the reduction of PGG2 to PGH2.[31],[32] 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.[33] 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.[34] 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.[35]

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.

Statistical analysis

Data were expressed as mean ± standard deviation of three individual experiments, and a P ≤ 0.05 was considered as statistically significant.


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}

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}{Figure 3}

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}{Figure 5}{Figure 6}{Figure 7}

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.


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.[2] 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[36],[37] but also provided insights into the use of alternative plant parts as Abhāvapratinidhi.[38]

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.[39] 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[40] and isolation of clerodendrin-A from the roots of both the species.[41] 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.


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.

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Conflicts of interest

There are no conflicts of interest.






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