Choline, a rediscovered Vitamin B4 that largely exists within the sort of phospholipids, plays a vital role in many biological functions in poultry. It is essential for building and maintaining cell membranes and organelles, like mitochondria and microsomes, and for bone maturation. It is also a necessary element of neurotransmitter acetylcholine, the foremost neurochemical within the nervous system concerned within the transmission of nerve impulses across synapses. The specialized structural feature of Choline chloride is its biologically active methyl groups through which it plays a significant role as a labile methyl group donor within the formation of methionine from homo-cysteine once being oxidised to betaine. Choline chloride helps in preventing abnormal accumulation of lipid and development of liver disease.

Unlike different vitamins, Choline is synthesised through de novo synthesis, however, the inability to form a decent quantity will lead to choline deficiency, causing growth retardation and perosis in young chicks. Moreover, the bioavailability of native choline varies mostly and depends on staple sources and bird-related factors like sort, strain, age, feed consumption, dietary crude macromolecule, and essential amino acid. Additionally, the supplementation of dietary methionine or different methyl group donors cannot fully replace the choline demand of chicks as avian species have restricted capability to hold out the initial step in choline synthesis, i.e., methylation of aminoethanol to methyl aminoethanol, that is contradictory to a situation with growing mammals like pigs or rats.
Hence, choline has become an essential feed additive within the ration of broiler chickens because the above-stated issues can be counteracted by adding enough amounts of synthetic choline chloride (SCC).

Choline chloride, a typical sort of Choline added to the animal diets, has some disadvantages like high hygroscopicity, acceleration of oxidative loss of vitamins, and the formation of trimethylamine (TMA) within the duct of the birds. TMA is a short-chain aliphatic amine formed from dietary Choline chloride in a reaction catalysed by microbial enzymes inside the gut. It is found in high concentrations in fish and is to blame for the characteristic odour of food. These drawbacks will influence the organic poultry production and hence, the utilization of Synthetic Choline Chloride in organic farming practices has been investigated.
Due to the importance of Choline chloride in poultry nutrition and production, the researchers have reinvestigated and explored the alternatives for artificial Choline chloride from natural sources. To counteract the drawbacks of Synthetic choline chloride addition, researchers have conjointly worked on the addition of herbal preparations in broiler rations. Numerous natural products and medicinal plants, together with crude extracts and compounds isolated from plants, are used as an alternate to Synthetic Choline Chloride in animal diets. Several researchers believe that these herbs will mimic the choline-like activity in poultry.

The polyherbal formulation (PHF), indexed as Kolin Plus and (M/s Natural Remedies Pvt Ltd, Bengaluru, India), is a combination of tree nilotica (A. nilotica) and herb (C. longa) belonging to the families of Mimosaceae and family Zingiberaceae, respectively. The hepatoprotective, inhibitor and lipophilic properties of extracts of those plants have been reported separately. However, there isn’t any scientific information demonstrating their Choline chloride-like activities in combined form in a choline deficiency model (CDM) in poultry.

There are not any applicable models for studying Choline chloride deficiency using weight gain as the major response criterion in broiler chickens. Similarly, many attempts to judge the importance of Choline chloride in poultry production have met with failure, as commercial ration had decent Choline chloride content and no distinction was ascertained with or without synthetic choline chloride. Therefore, an acceptable basal diet can permit the researchers to judge the issues of Choline chloride deficiency in broiler chickens.
SBM or soybean meal may be a principal supply of Choline chloride that satisfies the Choline chloride requirements in poultry diets. Therefore, once intact SBM is substituted by defatted SBM (i.e., soy macromolecule isolate [SPI]), a process to get rid of a part of the Choline, the sole distinction within the basal and Choline chloride-deficient diets would be the quantity of choline present. This modification overcomes the matter of adding contradictory factors aside from Choline chloride with the test ingredient.
Hence, the current study was planned to develop a CDM that might elicit the weight gain distinction in broiler chickens by feeding a basal diet with SBM or SPI. The induced CDM compared the impact of the newly developed PHF and SCC on growth performance, serum biochemistry, liver histopathology, and carcass traits of broiler chickens.
The study was designed to establish choline deficiency model (CDM) in broilers for evaluating efficacy of polyherbal formulation (PHF) in comparison with synthetic choline chloride (SCC). A total of 2,550 one-day-old Cobb 430 broiler chicks were randomly assigned to different groups in three experiments.
herbal analogues of Choline
In experiment 1, G1 and G2 served as normal controls and were fed a basal diet with 100% soybean meal (SBM) as a major protein source supplemented with and without SCC, respectively.
In G3, G4, G5, and G6 groups, SBM was replaced at 25%, 50%, 75%, and 100% by soy protein isolate (SPI) to induce a graded level of choline deficiency.
In experiment 2, PHF (500 and 1,000 g/ton) in comparison with SCC (1,000 g/ton) were evaluated.
In experiment 3, dose-response of PHF (200, 400, and 500 g/ton) with SCC (400 g/ton) was determined.
Replacement of SBM by SPI produced a linear decrease in body weight gain (BWG) with a poor feed conversion ratio (FCR). 25% SBM replacement by SPI yielded an optimum negative impact on BWG and FCR;
herbal analogues of Choline
herbal analogues of Choline
In experiment 2, PHF (500 and 1,000 g/ton) and SCC (1,000 g/ton) showed a similar performance in BWG, FCR and relative liver weight.
herbal analogues of Choline
In experiment 3, PHF produced an optimum efficacy at 400 g/ton and was comparable to SCC in the restoration of serum aspartate aminotransferase activity, abdominal fat, breast muscle lipid content and liver histopathological abnormalities.

Carcass characteristics: PHF vs SCC

herbal analogues of Choline
herbal analogues of Choline

Histopathological changes taking place in CDM

Replacement of SBM by SPI caused choline deficiency characterised by worsening of BWG, FCR, elevation in liver enzymes and histopathological changes indicating fatty liver. CDM was found valid for evaluating SCC and PHF.
In conclusion, choline deficiency could cause the growth of depression in meat broiler chickens. In this study, CDM was successfully induced and established using 75% SBM+25% SPI as a source of protein, which is suitable to measure the characteristic symptom of choline deficiency, i.e., growth performance in broiler chickens. CDM was found valid in the screening of products possessing choline-like activities. Also, it was confirmed that PHF has the potential to replace the function of 1 kg/ton of synthetic choline (choline chloride 60%) at 400 g/ton inclusion rate in broiler diets; this was reflected by the improved growth performance and feed efficiency.

For further reference kindly follow,

Evaluation of polyherbal formulation and synthetic choline chloride on choline deficiency model in broilers: implications on zootechnical parameters, serum biochemistry and liver histopathology Asian-Australas J Anim Sci. 2018 Nov; 31(11): 1795–1806.

Authors: Ramasamy Selvam, Marimuthu Saravanakumar, Subramaniyam Suresh, CV Chandrasekeran, and D’Souza Prashanth

Article by
Dr Ananth Krishna and Dr Mohita Gautam

Summary

Components of marketable herbal feed supplements are primarily evaluated based on their efficacy and safety to ensure quality. Nevertheless, as complex mixtures of different groups of primary and secondary metabolites, retention of overall phytochemical consistency with permissible levels of contaminants is critical to their efficacy and safety of herbal feed supplements. Herbs sourced from right geography, collected in the right season, authenticated appropriately and processed with stringent systems of manufacturing are serious factors to sustain efficacy with the consistency of phytochemicals inorder to manufacture herbal feed supplements. To ensure overall quality of herbal feed supplements through application of good agricultural and collection practice (GACP), good plant authentication and identification practice (GPAIP), good manufacturing practice (GMP) and good laboratory practice (GLP) in analysis are inevitable. In this communication, Natural Remedies Pvt. Ltd., recommends and adopts three steps process (HerbSecure) such as ensuring genuinity, safety, and efficacy to achieve the consistent quality of the herbal feed supplements to animal health industry.

Ensuring Genuinity

Quality of herbs used in the formulation of feed supplements start with sourcing. Sourcing should comply with Good Agricultural and Collection Practices (GACP). Main objectives of GACP are 1. Contribute to the quality assurance of plant materials used as the source for herbal feed supplements 2. To encourage and support the sustainable cultivation and collection of plants of good quality in ways that respect and support the conservation of herbs and the environment in general.  3. To provide traceability of the ingredients used in the formulation.

Good agricultural and collection practice starts with creating standard operating procedures (SOPs) for collection, post-harvest treatments like drying and maintaining traceability. Drawing a representative sample and equating with plant voucher herbarium specimens is the primary step in authenticity. Collected samples must be equated with a reference (standard) voucher specimens species-defining characters to ascertain the authenticity. An expert taxonomist is required to certify this step.

Other methods for plant authentication are macroscopic, microscopic character sets and their phytochemical (secondary metabolites) profiles. It is not unusual that most pharmacopeial monographs initiate the identification of herbs based on morphological and anatomical characters of dried and sorted plant parts (2). In phytochemical profile method, the chromatographic finger print of the sample developed using a validated analytical method is compared with a botanical reference material of the same species. The similarity of the finger printing of the sample to the reference material confirms the identity. Apart from that, presence of marker compounds which are specific to particular species also confirms identity (Figure 1).

Phytochemical profiling is extensively used for species and plant part specific identification since macroscopic and microscopic characters in dried or processed plant, parts are disturbed and creating authentication difficult. Phytochemical profiling chiefly employs high-performance thin-layer chromatography (HPTLC) and high-performance liquid chromatography in addition to gas chromatography (GC) (2, 4, 5). Among these techniques, thin-layer chromatography (TLC) and high-performance thin-layer chromatography (HPTLC) are widely used at Natural remedies Pvt. Ltd., for the generation of chemical fingerprints. In addition to above conventional methods, modern methods based on genomic profiling and DNA barcoding are complementary techniques to classical systematic approaches to plant authentication, which can be used for the unequivocal identification of plant species (6).

 

Figure 1. Methods used to determine the genuinity of herbs

Ensuring Safety:

Guaranteeing authentic medicinal herb with permissible impurities is the next important step towards accomplishing excellency in herbal products. Impurities include heavy metal, pesticide residues, and aflatoxins/mycotoxins. Heavy metal in plant species are accumulated from contaminated soils. More than 500 plant species are identified to store high levels of heavy metals, and in some cases, heavy metal occurrence in aerial parts exceed life-threatening toxicity levels to animals (2). Heavy metals (Pb, Hg, As, and Cd) are tested on herbal products using sophisticated techniques like atomic absorption spectroscopy (AAS), inductively coupled plasma mass spectrometry (ICP-MS) (Figure 2). The allowable limits for the heavy metals and metalloids in herb starting materials and preparations are prescribed by feed standard, WHO and individual herbal monographs of British and US pharmacopoeias (2).

Pesticide residues in medicinal herbs ascend from crop protection practices in cultivation. Pesticides broadly categorized to fungicides, insecticides, and weedicides. Multiple classes of pesticides like organochlorines, organophosphorus, carbamates, benzimidazoles, dithiocarbamates, and amino acid herbicides are used in plant cultivation. Quality driven herbal companies took the challenges of testing all the broad groups of pesticides using acceptable methods.

Other major contaminant that pose threat to safety of herbal products is mycotoxins. Out of hundreds of known mycotoxins, aflatoxins (Figure 2), ochratoxin A, fumonisins, zearalenone, and deoxynivalenol are the key ones. Mycotoxins are known to have neurotoxic, carcinogenic, immunotoxic, and teratogenic. Country specific feed standards, pharmacopoeias prescribe safe limits of aflatoxins and ochratoxin A in their documents. A number of validated methods of mycotoxin analysis including LC MS/MS GCMSMS (Figure 2) in the plant matrix are reported (7). Due to safety issues, it is critical to check fungal infection and prove that the herbal material is free of mycotoxin contamination especially after harvest and storage.

 

Figure 2. Detection of heavy metals and pesticide residues

In spite of the growing market demand on herbal products, there are still concerns associated with their safety. Surprisingly, not more than 10% of herbal products in the global market are actually standardized to known active components and strict quality control measures are not always wisely complied (8). For majority of these products in use, very little is known about their active and/or toxic constituents. This raises apprehension on their safety and consequences for their use as supplements. Toxicity testing using harmonized guidelines (Eg. ICH, OECD, schedule Y) in a Good Laboratory Practice (GLP) lab can divulge about risks which are associated with use of such herbs, consequently evading possible detrimental effects (9).

Ensuring Efficacy:

The success of modern analytical chemistry methods often conceals the problem since increasing amount of analytical data does not necessarily give more insight to biological effect. As alternative approaches, bioactivity-based detection of marker(s) can deliver valuable information about phytochemicals responsible for biological effects of complex materials such as herbal feed supplements. Effect directed analysis tries to interconnect instrumental analytical techniques with a biological/biochemical entity, which identifies or isolates substances of biological relevance (Figure 3). The connection of biological effects with the identification and quantification of phytochemicals leads to relevant answers to many questions (10).

It is utmost important to establish the synergy or additvity wherever more than one herb used in the supplement for the desired biological activity. Also, to confirm the efficacy it is vital that herbal product to undergo different levels of efficacy studies as mentioned in Figure 4. Once the bioactive molecule(s) identified as mentioned above it is important to determine the same in herbal products using qualitative and quantitative methods as it is equally important to verify the consistency across each batch using single or combination of analytical chemistry techniques (Figure 5) in the final product.

 

Figure 3. Bio-assay guided fractionation of plant (11)

Figure 4. Frame work of biological studies to develop herbal product

 

 

Figure 5. Verification of consistency between batches of poly herbal formulation

 

In conclusion, experience and knowledge on traditional and modern systems of herbal usage, infrastructure with sophisticated instruments, bioassays and able scientists, certified excellent process (GACP, GPAI, GMP, NABL, GLP, KOSHER, FAMIQS) would lead to delivering consistent, safe and cost effective herbal products to animal health industry.  The herbal feed supplements produced in Natural Remedies undergoes the fore said systems to ensure quality.

— Dr C V Chandrasekaran, M.V.Sc., Ph.D., Senior Manager, Research and Development, Natural Remedies Pvt. Ltd., Bangalore, India.

— Mr B. Murali, M.Sc., Assistant General Manager, Quality Control & Assurance, Natural Remedies Pvt. Ltd., Bangalore, India.

References:

  1. Hildreth J, Hrabeta-Robinson E, Applequist W, Betz J, Miller J. Standard operating procedure for the collection and preparation of voucher plant specimens for use in the nutraceutical industry. Anal Bioanal Chem. 2007, 389(1):13-17.
  2. Govindaraghavan S, Sucher NJ. Quality assessment of medicinal herbs and their extracts: Criteria and prerequisites for consistent safety and efficacy of herbal medicines. Review. Epilepsy Behav. 2015, 52:363-71.
  3. Sumner LW, Mendes P, Dixon RA. Plant metabolomics: large-scale phytochemistry in the functional genomics era. Review. Phytochemistry. 2003, 62:817-836.
  4. Reich E, Schibili A. High performance thin layer chromatography for the analysis of medicinal plants. New York: Thieme; 2007.
  5. Wagner H, Bauer R, Melchart D, Xiao P-G, Staudinger A. Chromatographic fingerprint analysis of herbal medicines. Wien: Springer-Verlag; 2011.
  6. Sucher NJ, Hennell JR, Carles MC. DNA fingerprinting, DNA barcoding, and next generation sequencing technology in plants. Methods Mol Biol 2012, 862:13–22.
  7. Selvi RC and Paramasivam M. Review on pesticide residue analytical methods and residue status in medicinal plants. Journal of Entomology and Zoology Studies 2017, 5: 945-950.
  8. Winston D, Maimes S. Adaptogens: Herbs for strength, stamina and stress relief. Rochester, Vermont: Healing Arts Press; 2007.
  9. Chandrasekaran CV, Sundarajan K, David K, Agarwal A. In vitro efficacy and safety of poly-herbal formulations. Toxicol In Vitro. 2010, 24:885-97.
  10. Weller MG. A unifying review of bioassay-guided fractionation, effect-directed analysis and related techniques. Sensors (Basel). 2012, 12:9181-91209.
  11. Deepak M, Dipankar G, Prashanth D, Asha MK, Amit A, Venkataraman BV. Tribulosin and beta-sitosterol-D-glucoside, the anthelmintic principles of Tribulus terrestris. Phytomedicine. 2002 9:753-756.

Summary

Components of marketable herbal feed supplements are primarily evaluated based on their efficacy and safety to ensure quality. Nevertheless, as complex mixtures of different groups of primary and secondary metabolites, retention of overall phytochemical consistency with permissible levels of contaminants is critical to their efficacy and safety of herbal feed supplements. Herbs sourced from right geography, collected in the right season, authenticated appropriately and processed with stringent systems of manufacturing are serious factors to sustain efficacy with the consistency of phytochemicals inorder to manufacture herbal feed supplements. To ensure overall quality of herbal feed supplements through application of good agricultural and collection practice (GACP), good plant authentication and identification practice (GPAIP), good manufacturing practice (GMP) and good laboratory practice (GLP) in analysis are inevitable. In this communication, Natural Remedies Pvt. Ltd., recommends and adopts three steps process (HerbSecure) such as ensuring genuinity, safety, and efficacy to achieve the consistent quality of the herbal feed supplements to animal health industry.

Ensuring Genuinity

Quality of herbs used in the formulation of feed supplements start with sourcing. Sourcing should comply with Good Agricultural and Collection Practices (GACP). Main objectives of GACP are 1. Contribute to the quality assurance of plant materials used as the source for herbal feed supplements 2. To encourage and support the sustainable cultivation and collection of plants of good quality in ways that respect and support the conservation of herbs and the environment in general.  3. To provide traceability of the ingredients used in the formulation.

Good agricultural and collection practice starts with creating standard operating procedures (SOPs) for collection, post-harvest treatments like drying and maintaining traceability. Drawing a representative sample and equating with plant voucher herbarium specimens is the primary step in authenticity. Collected samples must be equated with a reference (standard) voucher specimens species-defining characters to ascertain the authenticity. An expert taxonomist is required to certify this step.

Other methods for plant authentication are macroscopic, microscopic character sets and their phytochemical (secondary metabolites) profiles. It is not unusual that most pharmacopeial monographs initiate the identification of herbs based on morphological and anatomical characters of dried and sorted plant parts (2). In phytochemical profile method, the chromatographic finger print of the sample developed using a validated analytical method is compared with a botanical reference material of the same species. The similarity of the finger printing of the sample to the reference material confirms the identity. Apart from that, presence of marker compounds which are specific to particular species also confirms identity (Figure 1).

Phytochemical profiling is extensively used for species and plant part specific identification since macroscopic and microscopic characters in dried or processed plant, parts are disturbed and creating authentication difficult. Phytochemical profiling chiefly employs high-performance thin-layer chromatography (HPTLC) and high-performance liquid chromatography in addition to gas chromatography (GC) (2, 4, 5). Among these techniques, thin-layer chromatography (TLC) and high-performance thin-layer chromatography (HPTLC) are widely used at Natural remedies Pvt. Ltd., for the generation of chemical fingerprints. In addition to above conventional methods, modern methods based on genomic profiling and DNA barcoding are complementary techniques to classical systematic approaches to plant authentication, which can be used for the unequivocal identification of plant species (6).

 

Figure 1. Methods used to determine the genuinity of herbs

Ensuring Safety:

Guaranteeing authentic medicinal herb with permissible impurities is the next important step towards accomplishing excellency in herbal products. Impurities include heavy metal, pesticide residues, and aflatoxins/mycotoxins. Heavy metal in plant species are accumulated from contaminated soils. More than 500 plant species are identified to store high levels of heavy metals, and in some cases, heavy metal occurrence in aerial parts exceed life-threatening toxicity levels to animals (2). Heavy metals (Pb, Hg, As, and Cd) are tested on herbal products using sophisticated techniques like atomic absorption spectroscopy (AAS), inductively coupled plasma mass spectrometry (ICP-MS) (Figure 2). The allowable limits for the heavy metals and metalloids in herb starting materials and preparations are prescribed by feed standard, WHO and individual herbal monographs of British and US pharmacopoeias (2).

Pesticide residues in medicinal herbs ascend from crop protection practices in cultivation. Pesticides broadly categorized to fungicides, insecticides, and weedicides. Multiple classes of pesticides like organochlorines, organophosphorus, carbamates, benzimidazoles, dithiocarbamates, and amino acid herbicides are used in plant cultivation. Quality driven herbal companies took the challenges of testing all the broad groups of pesticides using acceptable methods.

Other major contaminant that pose threat to safety of herbal products is mycotoxins. Out of hundreds of known mycotoxins, aflatoxins (Figure 2), ochratoxin A, fumonisins, zearalenone, and deoxynivalenol are the key ones. Mycotoxins are known to have neurotoxic, carcinogenic, immunotoxic, and teratogenic. Country specific feed standards, pharmacopoeias prescribe safe limits of aflatoxins and ochratoxin A in their documents. A number of validated methods of mycotoxin analysis including LC MS/MS GCMSMS (Figure 2) in the plant matrix are reported (7). Due to safety issues, it is critical to check fungal infection and prove that the herbal material is free of mycotoxin contamination especially after harvest and storage.

 

Figure 2. Detection of heavy metals and pesticide residues

In spite of the growing market demand on herbal products, there are still concerns associated with their safety. Surprisingly, not more than 10% of herbal products in the global market are actually standardized to known active components and strict quality control measures are not always wisely complied (8). For majority of these products in use, very little is known about their active and/or toxic constituents. This raises apprehension on their safety and consequences for their use as supplements. Toxicity testing using harmonized guidelines (Eg. ICH, OECD, schedule Y) in a Good Laboratory Practice (GLP) lab can divulge about risks which are associated with use of such herbs, consequently evading possible detrimental effects (9).

Ensuring Efficacy:

The success of modern analytical chemistry methods often conceals the problem since increasing amount of analytical data does not necessarily give more insight to biological effect. As alternative approaches, bioactivity-based detection of marker(s) can deliver valuable information about phytochemicals responsible for biological effects of complex materials such as herbal feed supplements. Effect directed analysis tries to interconnect instrumental analytical techniques with a biological/biochemical entity, which identifies or isolates substances of biological relevance (Figure 3). The connection of biological effects with the identification and quantification of phytochemicals leads to relevant answers to many questions (10).

It is utmost important to establish the synergy or additvity wherever more than one herb used in the supplement for the desired biological activity. Also, to confirm the efficacy it is vital that herbal product to undergo different levels of efficacy studies as mentioned in Figure 4. Once the bioactive molecule(s) identified as mentioned above it is important to determine the same in herbal products using qualitative and quantitative methods as it is equally important to verify the consistency across each batch using single or combination of analytical chemistry techniques (Figure 5) in the final product.

 

Figure 3. Bio-assay guided fractionation of plant (11)

Figure 4. Frame work of biological studies to develop herbal product

 

 

Figure 5. Verification of consistency between batches of poly herbal formulation

 

In conclusion, experience and knowledge on traditional and modern systems of herbal usage, infrastructure with sophisticated instruments, bioassays and able scientists, certified excellent process (GACP, GPAI, GMP, NABL, GLP, KOSHER, FAMIQS) would lead to delivering consistent, safe and cost effective herbal products to animal health industry.  The herbal feed supplements produced in Natural Remedies undergoes the fore said systems to ensure quality.

— Dr C V Chandrasekaran, M.V.Sc., Ph.D., Senior Manager, Research and Development, Natural Remedies Pvt. Ltd., Bangalore, India.

— Mr B. Murali, M.Sc., Assistant General Manager, Quality Control & Assurance, Natural Remedies Pvt. Ltd., Bangalore, India.

References:

  1. Hildreth J, Hrabeta-Robinson E, Applequist W, Betz J, Miller J. Standard operating procedure for the collection and preparation of voucher plant specimens for use in the nutraceutical industry. Anal Bioanal Chem. 2007, 389(1):13-17.
  2. Govindaraghavan S, Sucher NJ. Quality assessment of medicinal herbs and their extracts: Criteria and prerequisites for consistent safety and efficacy of herbal medicines. Review. Epilepsy Behav. 2015, 52:363-71.
  3. Sumner LW, Mendes P, Dixon RA. Plant metabolomics: large-scale phytochemistry in the functional genomics era. Review. Phytochemistry. 2003, 62:817-836.
  4. Reich E, Schibili A. High performance thin layer chromatography for the analysis of medicinal plants. New York: Thieme; 2007.
  5. Wagner H, Bauer R, Melchart D, Xiao P-G, Staudinger A. Chromatographic fingerprint analysis of herbal medicines. Wien: Springer-Verlag; 2011.
  6. Sucher NJ, Hennell JR, Carles MC. DNA fingerprinting, DNA barcoding, and next generation sequencing technology in plants. Methods Mol Biol 2012, 862:13–22.
  7. Selvi RC and Paramasivam M. Review on pesticide residue analytical methods and residue status in medicinal plants. Journal of Entomology and Zoology Studies 2017, 5: 945-950.
  8. Winston D, Maimes S. Adaptogens: Herbs for strength, stamina and stress relief. Rochester, Vermont: Healing Arts Press; 2007.
  9. Chandrasekaran CV, Sundarajan K, David K, Agarwal A. In vitro efficacy and safety of poly-herbal formulations. Toxicol In Vitro. 2010, 24:885-97.
  10. Weller MG. A unifying review of bioassay-guided fractionation, effect-directed analysis and related techniques. Sensors (Basel). 2012, 12:9181-91209.
  11. Deepak M, Dipankar G, Prashanth D, Asha MK, Amit A, Venkataraman BV. Tribulosin and beta-sitosterol-D-glucoside, the anthelmintic principles of Tribulus terrestris. Phytomedicine. 2002 9:753-756.

ABSTRACT

Background: Curcuma longa has long history of medicinal use in Ayurveda. A unique product NR‑INF‑02 was prepared from C. longa that was standardized to contain turmerosaccharides. Objective: The present study investigated the effect of turmerosaccharides rich fraction of NR‑INF‑02 on monosodium iodoacetate (MIA)‑induced OA pain animal model that mimics human OA. Further, the analgesic effect of turmerosaccharides rich fraction was compared to turmerosaccharides less fraction of NR‑INF‑02. Materials and Methods: OA pain was chemically induced by intra‑articular administration of single dose of 25 µl of 0.9% saline containing 0.3 mg MIA into the right knee of male albino Wistar rat. Turmerosaccharides rich fraction and turmerosaccharides less fraction (at 22.5, 45 and 90 mg/kg rat body weight dose levels) were administered as a single dose orally on day 5 of post‑MIA injection. OA pain was measured using hind limb weight‑bearing ability at 1, 3, 6, and 24 h post-test substance administration on day 5. Results: Oral administration of turmerosaccharides rich fraction and turmerosaccharides less fraction (at 45 and 90 mg/kg) although significantly decreased the OA pain at all the intervals, the effect of turmerosaccharides rich fraction (57%) on OA pain was superior to turmerosaccharides less fraction (35%). Conclusion: Bioactive turmerosaccharides from C. longa extract contribute to the observed anti‑arthritic effect in rats.

Key words: Analgesia effect, Curcuma longa, monosodium iodoacetate, NR‑INF‑02, osteoarthritis, turmerosaccharides

SUMMARY

  • Osteoarthritic pain was induced by intra-articular injection of MIA into the right knee
  • Single administration of TRF/TLF on day 5 resulted in dose-dependent significant reduction of OA pain
  • TRF showed better analgesic activity than TLF
  • TRF at 45 and 90 mg/kg has similar effects on OA pain as that of tramadol

Turmerosaccharides identified as bioactive constituents of C. longa extract.

Abbreviations used: MIA: Monosodium iodoacetate; i.ar: Intra-articular; OA: Osteoarthritis; TRF: Turmerosaccharides rich fraction; TLF: Turmerosaccharides less fraction; PGE2: Prostaglandin E2; ROS: Reactive oxygen species.

Correspondence:
Dr. Chandrasekaran Chinampudur Velusami,
Department of Biology, R & D Centre, Natural
Remedies Private Limited, 5B, Veerasandra Indl. Area,
19th K. M. Stone, Hosur Road, Electronic City Post,
Bengaluru ‑ 560 100, Karnataka, India.
E‑mail: cvc@naturalremedy.com
DOI: 10.4103/pm.pm_465_16
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Website: www.phcog.com
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INTRODUCTION

Turmeric (Curcuma longa) belonging to Zingiberaceae family has a very long history of culinary, cosmetic, and medicinal use. In Ayurveda practices, turmeric is well‑documented to have several medicinal properties for the treatment of various respiratory conditions (e.g., asthma, bronchial hyperactivity, and allergy) as well as for liver disorders, anorexia, rheumatism, diabetic wounds, runny nose, cough, sinusitis, sprains, inflammation, and swelling. While in traditional Chinese medicine, it is used to treat diseases associated with abdominal pain.[1,2] Thus, there is substantial evidence in traditional system of medicine pertinent to the effectiveness of turmeric against inflammation and pain.Ayurvedic herbal medicine preparation methods as mentioned in classical Ayurvedic books, namely, Bhaiṣajya kalpanā vijñānam and Śā̄rṅgadhara Saṃhitā̄ of Śā̄rṅgadharā̄cā̄rya, include dosage forms such as svarasa (juice), kalka (bolus/paste), kvatha (decoction), hima (cold infusion), phaṇṭa (hot infusion), and curna (powder). The solvents generally used for the traditional herbal preparation include water,

Ayurvedic herbal medicine preparation methods as mentioned in classical Ayurvedic books, namely, Bhaiṣajya kalpanā vijñānam and Śā̄rṅgadhara Saṃhitā̄ of Śā̄rṅgadharā̄cā̄rya, include dosage forms such as svarasa (juice), kalka (bolus/paste), kvatha (decoction), hima (cold infusion), phaṇṭa (hot infusion), and curna (powder). The solvents generally used for the traditional herbal preparation include water, ghee/oil, and milk. This clearly provides evidence that organic solvent extraction is not defined in Ayurveda for most of the herbal medicines and possibly the water/aqueous extraction procedures yield herbal medicines that are efficacious in treating diseases/disorders.[3] In addition, few scientific reports on the pharmacological activities such as antioxidant, anti‑tumor, anti‑diabetic, immune modulatory and anti‑depressant activity of aqueous extracts of C. longa are available.


This is an open access article distributed under the terms of the Creative Commons Attribution‑NonCommercial‑ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non‑commercially, as long as the author is credited and the new creations are licensed under the identical terms.

For reprints contact: reprints@medknow.com

Cite this article as: Bethapudi B, Murugan S, Illuri R, Mundkinajeddu D, Velusami CC. Bioactive turmerosaccharides from Curcuma longa extract (NR-INF-02): Potential ameliorating effect on osteoarthritis pain. Phcog Mag 0;0:0.


In view of the above, a formulation was developed from C. longa using the decoction method with water as solvent and was termed as NR‑INF‑02 or Turmacin™. NR‑INF‑02 was standardized to contain turmerosaccharides (>10% w/w) and negligible amount of curcuminoids.[8]

NR‑INF‑02 rich in turmerosaccharides was extensively studied in in vitro and in vivo safety and efficacy studies. NR‑INF‑02 demonstrated significant anti‑inflammatory activity in acute (carrageenan and xylene) and chronic (cotton pellet granuloma) inflammatory in vivo models.[9] In addition, NR‑INF‑02 was fractionated into turmerosaccharides rich fraction and turmerosaccharides less fraction. These fractions were further assessed for anti‑inflammatory activity and safety. In vitro and in vivo studies indicated that anti‑inflammatory activity of turmerosaccharides rich fraction of NR‑INF‑02 was superior to turmerosaccharides less fraction.[8] Further, the LD50 was found to be >5000 mg/kg rat body weight in acute oral toxicity – fixed dose procedure using the OECD test guideline No. 420.[10] Thus, these studies clearly indicated that the possible anti‑inflammatory activity of NR‑INF‑02 is primarily due to turmerosaccharides. However, the turmerosaccharides rich fraction was not investigated in in vivo monosodium iodoacetate (MIA)‑induced osteoarthritic (OA) pain model.Hence, the present study was conducted to investigate if turmerosaccharides fraction of NR‑INF‑02 contributes for relieving pain in an MIA‑induced OA rat model, which mimics the pain and changes associated with human OA. In addition, the study also compared the analgesic effects of turmerosaccharides rich fraction and turmerosaccharides less fraction of NR‑INF‑02.

Hence, the present study was conducted to investigate if turmerosaccharides fraction of NR‑INF‑02 contributes for relieving pain in an MIA‑induced OA rat model, which mimics the pain and changes associated with human OA. In addition, the study also compared the analgesic effects of turmerosaccharides rich fraction and turmerosaccharides less fraction of NR‑INF‑02.

MATERIALS AND METHODS Animals

Male albino Wistar rats bred at central animal facility, Research and Development Center, Natural Remedies Private Limited, were acclimatized for 5 days before experimentation. Rats weighing 240–250 g at the start of experimentation were kept under optimal temperature (25 ± 2°C) and 30%–70% relative humidity. Rats were provided access to rodent feed pellets (VRK Nutritional Solutions) and ultraviolet purified water ad libitum. All the animal procedures were approved by the Institutional Animal Ethics Committee of Natural Remedies, Bengaluru.

Preparation of test substances

Fractionation of NR‑INF‑02 into turmerosaccharides rich fraction and turmerosaccharides less fraction has been described in our earlier publication.[8] In brief, NR‑INF‑02 was dissolved in water, and 5 volumes ethanol was added. The mixture was centrifuged for 20 min at 2000 rpm. The precipitate obtained after centrifugation was stirred with 5 volumes of ethanol at room temperature for 10 min and filtered. After filtration, the retentate obtained was dried under vacuum at <70°C to obtain turmerosaccharides rich fraction. While the supernatant obtained was concentrated under vacuum to obtain turmerosaccharides less fraction.

Experimental procedure

The animals were randomly assigned into nine groups as G1 = Normal/vehicle control, G2 = MIA control, G3 = Tramadol at 10 mg/kg as a reference control, G4–G6 = Turmerosaccharides rich fraction at 22.5, 45, and 90 mg/kg dose levels, respectively, and G7–G9 = Turmerosaccharides less fraction at 22.5, 45, and 90 mg/kg dose levels, respectively. Each group was assigned six rats that were housed two in number per cage. Individual rats were identified by cage card and markings.

OA was induced by intra‑articular (i.ar) injection of MIA (Sigma‑Aldrich, USA) solution into the knee joint. Anesthetized rats of all the groups except normal/vehicle control received single dose of 25 µl of 0.9% saline containing 0.3 mg of MIA into the i.ar space of the right knee through the infrapatellar ligament. While left knee was injected with 25 µl of 0.9% saline only. While normal/vehicle control rats received 25 µl of 0.9% saline into the right and left knee joints. MIA and saline were freshly prepared under sterile conditions and were injected into the knee using 27‑gauge needle into the joint space.[11]

Tramadol at one dose level, turmerosaccharides rich fraction and turmerosaccharides less fraction at three dose levels were administered to the respective groups as a single oral administration on day 5 post‑MIA injection. Analgesic effects of the test substances were measured on day 5 at various time intervals (1, 3, 6, and 24 h).

Assessment of pain behavior

A hind limb static weight‑bearing incapacitance tester (Bioseb, France) was used to assess the pain behavior. Pain associated with OA is characterized by hind limb weight‑bearing distribution asymmetry. The difference in the distribution of weight on the left and the right hind limbs was measured as an objective measurement of pain.

All the groups were evaluated for hind limb weight bearing on day 0, 1, 3, and 5 (1, 3, 6, and 24 h post-test substance administration on day 5). While the analgesic effects of tramadol, turmerosaccharides rich fraction and turmerosaccharides less fraction was measured at 1, 3, 6, and 24 h post-test substance administration on day 5 following MIA injection. Animals were placed into a holder where the animal was comfortably maintained while their hind paws rest on two separate sensor plates. The animal was allowed to become accustomed to the apparatus. When stationary, the force exerted on the plate by each hind paw was recorded over a period and the same is expressed in grams. Four–five consecutive 5‑s readings were taken and averaged to obtain the mean score. Results were expressed as difference between hind paw weight distribution, percentage weight born, and area under curve (AUC) from the time of test substance administration to 24 h post-test substance administration.

Statistical analysis

The difference between hind paw weight distribution values was expressed as mean ± standard error mean. Data were analyzed using one‑way ANOVA followed by Bonferroni’s post hoc test for multiple group comparison. If error variance was found to be heterogenous, logarithmic transformation of raw data was performed and analyzed accordingly. Values of P ≤ 0.05 were considered statistically significant. Data were processed using statistical software IBM SPSS version 21. AUC was calculated using GraphPad prism version 5, GraphPad software Inc., California, USA.

RESULTS

Effect of oral administration of turmerosaccharides rich fraction and turmerosaccharides less fraction on monosodium iodoacetate‑induced osteoarthritic pain

Intra-articular injection of MIA (0.3 mg in 25 µl of 0.9% saline) into the right knee‑induced OA pain. OA pain resulted in statistically significant reduction on weight bearing on the right hind limb as compared to normal control rats indicating increased pain response. Single oral administration of turmerosaccharides rich fraction or turmerosaccharides less fraction on day 5 resulted in dose‑dependent significant reduction of OA pain response induced by MIA.

The difference in the weight distribution between left and right hind limbs (left‑right) was significantly reduced in tramadol, turmerosaccharides rich fraction (45 and 90 mg/kg), and turmerosaccharides less fraction (45 and 90 mg/kg)‑treated groups at all the time intervals (1, 3, 6 and 24 h) as compared to MIA‑treated group. However, turmerosaccharides rich fraction showed superior activity on OA pain when compared to turmerosaccharides less fraction [Figure 1].

The total AUC for difference in weight distribution (left and right hind limbs) was calculated from 0 to 24 h post-test substance administration. MIA‑treated group demonstrated a significant increase in AUC as compared to normal control group. While the AUC for tramadol, turmerosaccharides rich fraction, and turmerosaccharides less fraction‑treated groups were significantly reduced as compared to MIA‑treated group indicating reduction in OA pain over a period of 24 h [Figure 2].

Comparison of tramadol‑treated group with turmerosaccharides rich fraction and turmerosaccharides less fraction‑treated groups were

Figure 1: Effect of oral administration of turmerosaccharides rich fraction and turmerosaccharides less fraction at day 5 after monosodium iodoacetate (0.3 mg in 25 µl) intra‑articular injection. The effect on osteoarthritic pain evaluated at 1, 3, 6, and 24 h following oral administration of test substances. Data were expressed as difference in weight distribution on hind limbs (left‑right). Each value is mean ± standard error mean (n = 6) of four to five experiments. *P < 0.05 significantly different from normal control group. #P < 0.05 significantly different from monosodium iodoacetate control group

performed. There was no significant difference between tramadol treatment and turmerosaccharides rich fraction (90 mg/kg) treatment on OA pain at all intervals (1, 3, 6, and 24 h) evaluated on day 5. Whereas no significant difference between tramadol and turmerosaccharides rich fraction (45 mg/kg) treatment on OA pain was observed at all the intervals evaluated except for the first hour post treatment. Thus, turmerosaccharides rich fraction at 45 and 90 mg/kg has similar effects on OA pain as that of tramadol. Turmerosaccharides rich fraction was compared with turmerosaccharides less fraction‑treated groups with their corresponding dose levels. Statistical analysis indicated that at all intervals turmerosaccharides rich fraction 90 mg/kg was significantly more effective on OA pain over turmerosaccharides less fraction 90 mg/ kg [Table 1].

The effect of oral administration of test substance was also calculated as percentage inhibition of pain with respect to MIA control group. Tramadol demonstrated maximal pain reduction by 55%, 6 h postadministration. The turmerosaccharides rich fraction at 45 mg/kg dose level showed maximal pain reduction by 50%, 6 h postadministration. Correspondingly, turmerosaccharides less fraction at 45 mg/kg dose level showed maximal pain reduction by

Figure 2: Total area under the curve for difference in weight distribution on hind limbs from 0–24 h on day 5. Results are expressed as mean ± standard error mean 9 (n = 6). *P < 0.05 significantly different from normal control group. #P < 0.05 significantly different from monosodium iodoacetate control group

Table 1: Multiple comparison of the effects of tramadol, turmerosaccharides rich fraction, and turmerosaccharides less fraction at day 5 after monosodium iodoacetate injection

Group
Change in hind paw weight distribution (g)
Day 5/0 h Day 5/1 h Day 5/3 h Day 5/6 h Day 5/24 h
Normal control 10 ml/kg 0.5% CMC p.o (25 µl saline i.ar) 1.11±0.39 1.12±0.29 1.42±0.41 0.48±0.15 0.85±0.18
MIA 0.3 mg 10 ml/kg 0.5% CMC p.o (0.3 mg in 25 µl saline i.ar) 35.33±0.31* 33.55±0.55* 31.93±1.02* 34.80±0.92* 31.91±0.86*
Tramadol 10 mg/kg p.o (0.3 mg MIA in 25 µl saline i.ar) 33.69±0.70 21.40±0.74 16.04±1.21 15.70±0.69 16.96±1.30
TRF 22.5 mg/kg p.o (0.3 mg MIA in 25 µl saline i.ar) 34.31±0.84 27.01±0.33a 28.33±2.19a 22.01±0.39a 24.57±0.66a
TRF 45 mg/kg p.o (0.3 mg MIA in 25 µl saline i.ar) 34.51±0.89 26.25±0.33a 20.37±0.84 17.39±0.21 20.55±0.28
TRF 90 mg/kg p.o (0.3 mg MIA in 25 µl saline i.ar) 36.06±0.42 24.28±0.36 16.68±0.26 14.88±0.30 16.17±0.47
TLF 22.5 mg/kg p.o (0.3 mg MIA in 25 µl saline i.ar) 34.25±0.51 29.63±0.20a,b 27.50±0.55a 27.29±1.30a 27.31±0.98a
TLF 45 mg/kg p.o (0.3 mg MIA in 25 µl saline i.ar) 35.17±0.40 27.17±0.97a 24.82±0.96a 24.80±0.32a,c 26.47±0.55a,c
TLF 90 mg/kg p.o (0.3 mg MIA in 25 µl saline i.ar) 35.81±1.31 26.27±1.25 25.18±0.27a,d 22.51±0.64a,d 23.55±1.06d

Data were expressed as difference in weight distribution on hind limbs (left‑right). Each value is mean±SEM (n=6). *P<0.05 significantly different from normal control group; aP<0.05 tramadol versus TRF and TLF; bP<0.05 TRF (22.5 mg/kg) versus TLF (22.5 mg/kg); cP<0.05 TRF (45 mg/kg) versus TLF (45 mg/kg); dP<0.05 TRF (90 mg/kg) versus TLF (90 mg/kg). i.ar: Intra‑articular; CMC: Carboxymethylcellulose; MIA: Monosodium iodoacetate; SEM: Standard error of mean; TRF: Turmerosaccharides rich fraction; TLF: Turmerosaccharides less fraction

Table 1: Multiple comparison of the effects of tramadol, turmerosaccharides rich fraction, and turmerosaccharides less fraction at day 5 after monosodium iodoacetate injection

Groups Percentage reduction of pain severity (%)
Day 5/1 h Day 5/3 h Day 5/6 h Day 5/24 h
Tramadol 10 mg/kg p.o (0.3 mg MIA in 25 µl saline i.ar) 36.21 49.77 54.89 46.84
TRF 22.5 mg/kg p.o (0.3 mg MIA in 25 µl saline i.ar) 19.48 11.25 36.74 23.00
TRF 45 mg/kg p.o (0.3 mg MIA in 25 µl saline i.ar) 21.75 36.18 50.02 35.60
TRF 90 mg/kg p.o (0.3 mg MIA in 25 µl saline i.ar) 27.63 47.75 57.24 49.33
TLF 22.5 mg/kg p.o (0.3 mg MIA in 25 µl saline i.ar) 11.69 13.86 21.58 14.42
TLF 45 mg/kg p.o (0.3 mg MIA in 25 µl saline i.ar) 19.01 22.26 28.74 17.05
TLF 90 mg/kg p.o (0.3 mg MIA in 25 µl saline i.ar) 21.69 21.13 35.33 26.18

29%, 6 h postadministration. While turmerosaccharides rich fraction at 90 mg/kg dose level showed maximal pain reduction by 57%, 6 h postadministration. Correspondingly, turmerosaccharides less fraction at 90 mg/kg dose level demonstrated maximal pain reduction by 35%, 6 h postadministration [Table 2].

DISCUSSION

OA is the most common form of joint disease affecting over one‑half of people older than 65 years of age. OA primarily affects weight‑bearing joints such as knee and hip. Currently, acetaminophen, nonsteroidal anti‑inflammatory drug, opioids, and corticosteroids are used for treating OA pain however they are associated with adverse effects.[12,13] For these reasons, new agents with ability to attenuate OA‑associated pain and improve joint function are a welcome addition. In addition, the penchant toward natural products as a safe alternative to current pharmacological therapies[14] also promulgated to develop NR‑INF‑02 for OA pain. NR‑INF‑02 is prepared from polar fraction of C. longa and is standardized to contain turmerosaccharides.

In recent times, research has been focused on understanding the role of phytoconstituents such as flavonoids, polyphenols in modulating OA pain.

  • Hence, the present study has investigated whether turmerosaccharides fraction of NR‑INR‑02 contributes for relieving OA pain. In addition, the study also compared the effects of turmerosaccharides rich fraction and turmerosaccharides less fractions of NR‑INR‑02 on OA pain.

The effects of turmerosaccharides rich fraction and turmerosaccharides less fractions on OA pain were investigated in MIA‑OA pain model. MIA is a metabolic inhibitor; injection of MIA into the joints inhibits glyceraldehydes‑3‑phospate dehydrogenase activity in chondrocytes, resulting in disruption of glycolysis and eventual cell death. This model has been well established to test pharmacological agents for their ability to treat OA pain and mimics behavioral (pain), pathologic, and pharmacologic features associated with human OA.[12] Hence, attenuation of MIA‑OA pain by turmerosaccharides rich fraction directly connotes its effect on OA pain. In addition, it gives indication that turmerosaccharides may contribute to effect of NR‑INF‑02 on OA pain.

MIA injection into the right hind paw in the present study resulted in a significant decrease on weight bearing in the right hind paw. When administered at 0.3 mg per joint, the change in weight distribution was found to reach a maximum level on day 5 and maintained the level for 21 days (unpublished observations). Hence, the turmerosaccharides rich fraction and turmerosaccharides less fractions were administered on day 5 when the pain behavior reached maximal level and were assessed for analgesic effects. The results of the study indicated that turmerosaccharides rich fraction and turmerosaccharides less fractions administered orally produced significant analgesic activity. However, the analgesic effect produced by turmerosaccharides rich fraction was

superior to turmerosaccharides less fraction. Also, duration of action of turmerosaccharides rich fraction was higher than turmerosaccharides less fraction. We observed that 57% of analgesic activity was exhibited by turmerosaccharides rich fraction while turmerosaccharides less fraction produced 35% of analgesic activity. Thus, the current study findings indicate that turmerosaccharides are the major phytoactives that contribute to the analgesic activity of NR‑INF‑02. These observations are in agreement with the findings of Chandrasekaran et al. 2013[8] and Illuri et al. 2015[10] These two studies indicated that turmerosaccharides of NR‑INF‑02 contributed to the anti‑inflammatory effects. The analgesic effect of turmerosaccharides rich fraction appears to be similar to the anti‑nociceptive activity of synthetic drugs, tramadol that resulted in 55% of anti‑nociceptive activity.[15] Hence, the present study clearly indicates that turmerosaccharides are the phytoactives responsible for analgesic activity of NR‑INF‑02.

Induction of several factors such as NF-kβ, inflammatory mediators, eicosanoids and cytokines are responsible for OA pain.[15,16] The inflammatory mediators released following MIA injection cause acute synovial inflammation. As the pain fibers are present in synovium, ligaments, bone, muscle, and meniscus of knee, acute synovial inflammation induced by MIA causes peripheral and central sensitization. The process of sensitization is thought to be the basis of OA pain.[17] The turmerosaccharides of NR‑INF‑02 may exert its analgesic activity on the MIA‑induced model of OA pain through its anti‑inflammatory activity. The turmerosaccharides were shown to have anti‑inflammatory activity as it inhibited PGE2[8] that contribute to structural damage of knee joint, and subsequently, OA pain. Hence, PGE2 would have contributed to analgesic effect.

In addition, MIA also induces chondrocytes apoptosis primarily through ROS and ROS generation results in inflammation of articular cartilage.[18] Thus, anti‑oxidant ability or the oxidative defences will decrease chondrocyte damage. The turmerosaccharides from C. longa demonstrated marked radical scavenging activity in in vitro assays[19] Hence, the anti‑oxidant effects of turmerosaccharides would have resulted in scavenging ROS and thus resulted in chondrocyte protection and further analgesic activity. Thus, modulation of inflammatory mediators and chondrocyte protection would have contributed to the activity of turmerosaccharides rich fraction on OA pain.

Eventhough, turmerosaccharides less fraction showed significant analgesic effect, it is inferior to turmerosaccharides rich fraction. This indicates that turmerosaccharides less fractions might contain phytoactives responsible for analgesic effect. As indicated by Ramadas and Srinivas[20] and Angel et al.[21] water extract of C. longa contains glycoprotein, which might have contributed to the observed effect on OA pain. However, the phytoactives of turmerosaccharides less fraction are yet to be explored. Further, the mechanism of action of turmerosaccharides at molecular level are yet to be explored.

CONCLUSION

The current study demonstrated that turmerosaccharides rich fraction attenuated MIA‑OA pain. Thus, the study findings suggest that turmerosaccharides remain the major phytochemical actives of C. longa (NR-INF-02) in decreasing the OA pain.

Financial support and sponsorship

The authors are thankful to Indo‑Spanish Joint Programme, Department of Biotechnology of India, Centre for the development of Industrial Technology of Spain for providing partial financial assistance to this study.

Conflicts of interest

There are no conflicts of interest.

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A randomized, double-blind, placebo-controlled study was conducted to evaluate the efficacy of OciBest, an extract of Ocimum tenuiflorum Linn. in symptomatic control of general stress. The participants received either placebo (n = 79) or OciBest (n = 71; 1200 mg of actives per day) for six weeks. The severity of stress-related symptoms was self-evaluated by patients at weeks 0, 2, 4 and 6 of the trial period using a symptom rating scale. After six weeks of intervention, scores of symptoms such as forgetfulness, sexual problems of recent origin, frequent feeling of exhaustion, and frequent sleep problems of recent origin decreased significantly (P ≤ 0.05) in OciBest group as compared with placebo group. Also, the total symptom scores of OciBest group revealed significant reduction (P ≤ 0.05) as compared to placebo group. The overall improvement in Oci

A. J. Joshua, K. S. Goudar, N. Sameera, G. P. Kumar, B. Murali, N. Dinakar and A. Amit

DOI : 10.3844/ajptsp.2010.42.47

American Journal of Pharmacology and Toxicology

Volume 5, Issue 1

Pages 42-47

Abstract

Problem statement: Herbal remedies form one of the effective strategies for management of livestock healthcare. Despite the availability of extensive pharmacological information, the toxicological data on herbs and herbal preparations seem to be scanty. The objective of the present investigation was to evaluate the acute oral toxicity of some herbal veterinary preparations in albino Wistar rats. Approach: In the sighting study, the investigational substances (Rumbion

Copyright

© 2010 A. J. Joshua, K. S. Goudar, N. Sameera, G. P. Kumar, B. Murali, N. Dinakar and A. Amit. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.