HERBAL DRUG STANDARDIZATION
HARISH KAKRANI* & PURVI H. KAKRANI **
* CVM INSTITUTE OF PHARMACY, ADIT CAMPUS, NEW VALLABH VIDYANAGAR
Herbal Drug standardization
Herbal formulations are the remedies which cure the diseases rather than alleviating the symptoms. They are natural, safe, and of lower cost; consumers worldwide are spending more out-of-pocket money on them. Herbal drug technology includes all the steps that are involved in converting botanical materials into medicines. Numerous drugs have entered the international market through exploration of ethnopharmacology and traditional medicine.
The increasing reports of adverse reactions, has drawn the attention of many regulatory agencies for the standardization of herbal formulations. Unlike conventional pharmaceutical products, herbal medicinal products may vary in composition and properties, which are usually prepared from synthetic, chemically pure materials by means of reproducible manufacturing techniques and procedures. Correct identification and quality assurance of the starting material is, therefore, an essential prerequisite to ensure reproducible quality of herbal medicine, which contributes to its safety and efficacy. Standardization is a system that ensures that every dosage form that is being sold has a predefined amount of quantity & quality of ingredients and will induce its intended therapeutic effect.
Conventional methods for standardization of herbal formulation
Most of the regulatory guidelines and pharmacopoeias suggest macroscopic and microscopic evaluation and chemical profiling of the botanical materials for quality control and standardization7–9. Thin layer chromatography (TLC) and high performance thin layer chromatography (HPTLC) are routinely used as valuable tools for qualitative determination of small amounts of impurities. In addition, many analytical techniques such as volumetric analysis, gravimetric determinations, gas chromatography, column chromatography, high performance liquid chromatography and spectrophotometric methods are also frequently used for quality control and standardization. However, these parameters are judged subjectively and substitutes or adulterants may closely resemble the genuine material.
Difficulties in conventional approaches:
The use of chromatographic techniques and marker compounds to standardize botanical preparations has limitations because of their variable sources and chemical complexity. Variability in the flavors, aroma and physical characteristics of wine and coffee from year to year and region to region, provide a good analogy. Many factors may affect the ultimate chemical profile of any herb. Intrinsic factors such as genetics and extrinsic factors such as cultivation, harvesting, drying and storage conditions are a few examples. Routine chemotaxonomic studies provide only a qualitative account of secondary metabolites. For quantitative studies, use of specific markers that can be easily analyzed to distinguish between varieties, remains a preferred option. Such metabolites being used as markers may or may not be therapeutically active, but should ideally be neutral to environmental effects and management practices.
In order to ensure efficacy, selection of the correct chemotype of the plant is necessary. Even when there are many known chemotypes of a plant species, selection of the right chemotype to which clinical effects are attributed is difficult. For example, Withania somnifera is reported to have three chemotypes depending upon the presence of a class of closely related steroidal lactones like withanolides, withaferin A, etc. The content of withanolides, withaferin A and other biologically active compounds may vary depending upon the environment, genotype, time of collection of plant material, etc. Hence selection of the right chemotype having therapeutic efficacy is important. Another difficulty encountered in the selection of the correct plant material is to establish the identity of certain species that may be known by different binomial botanical names in different regions. For example, Shankhapushpi, which is an important ‘medhya rasayan’ in ayurveda is equated with any one of the following plants depending upon the region in India: Canscora decussata, Evolvulus alsinoides and Clitoria ternata. Certain rare and expensive medicinal plant species are often adulterated or substituted by morphologically similar, easily available or less expensive species. For example, Swertia chirata is frequently adulterated or substituted by the cheaper Andrographis paniculata. In view of these limitations there is need for a new approach that can complement or, in certain situations, serve as an alternative. Some of the newly emerging techniques for ensuring correct botanical identity and quality include HerboprintTM, which in addition to chemoprofile also considers ayurvedic properties, and capillary electrophoresis which is a faster, precise and sensitive method and has recently been used to ascertain the botanical identity and quality of Ephedrae herbal, Coptidis rhizoma, Ginseng radix and Paeoniae radix.
DNA finger printing technique:
This concept of fingerprinting has been increasingly applied in the past few decades to determine the ancestry of plants, animals and other microorganisms. DNA fingerprinting of herbal drugs, though still in its early years, seems to be a promising tool for the authentication of medicinal plant species and for ensuring better quality herbs and nutraceuticals. This is especially useful in case of those that are frequently substituted or adulterated with other species or varieties that are morphologically and/or phytochemically indistinguishable.
Additional motivation for using DNA fingerprinting on commercial herbal drugs is the availability of intact genomic DNA from plant samples after they are processed. Adulterants can be distinguished even in processed samples, enabling the authentication of the drug. Studies have reported the genotyping of several medicinal plants, and have made available their DNA fingerprints. However these results should be taken with a grain of salt as the plants are often sourced from a variety of locations through the world.
Another important issue is that DNA fingerprint will remain the same irrespective of the plant part used, while the phytochemical content will vary with the plant part used, physiology and environment. DNA fingerprinting ensures presence of the correct genotype but does not reveal the contents of the active principle or chemical constituents. Hence DNA analysis and pharmacognostic techniques for chemoprofiling such as TLC, HPTLC, etc. will have to be used hand in hand rather than in isolation. Identification of quantitative-trait loci34 that are closely linked to a biologically active phytochemical will prove to be useful. Several attempts have been made in recent years, to correlate DNA markers with qualitative and quantitative variations in phytochemical composition among closely related species87–92. Proper integration of molecular techniques and analytical tools will lead to the development of a comprehensive system of botanical characterization that can be conveniently applied at the industry level for quality control of botanicals. Ayurvedic classification of medicinal plant is based on basic principles and therapeutic characters that may have a genetic basis.
DNA markers, which may have several advantages over typical phenotype markers
As the genetic composition is unique for each species and is not affected by age, physiological conditions as well as environmental factors. DNA can be extracted from fresh or dried organic tissue of the botanical material; hence the physical form of the sample for assessment does not restrict detection.
The varying drug content of different species of herbal plants has been a problem in the production of standardized herbal medicines, where a particular plant from a region can be linked to a specific drug content and thus have a therapeutic value assigned to it, even though similar plants from another region may not share the same levels of the drug. Factors such as soil, climate and adaptability dictate the viability of a particular species and subsequently its drug content. In such cases, there are observed variations in the genetic composition of the plant, in addition to varying amounts of the active drug compound. When used commercially, two factors affect the final drug quality:
The variability with respect to strain-specific drug content.
The potential adulteration of plant drugs with extracts from plants that have lower drug content.
Types of DNA markers used in plant genome analysis
Various types of DNA-based molecular techniques are utilized to evaluate DNA polymorphism. These are hybridization-based methods, polymerase chain reaction (PCR)-based methods and sequencing-based methods.
Hybridization-based methods include restriction fragment length polymorphism (RFLP) and variable number tandem repeats. Labeled probes such as random genomic clones, cDNA clones, probes for microsatellite and minisatellite sequences are hybridized to filters containing DNA, which has been digested with restriction enzymes. Polymorphisms are detected by presence or absence of bands upon hybridization.
PCR-based markers involve in vitro amplification of particular DNA sequences or loci, with the help of specific or arbitrary oligonucleotide primers and the thermostable DNA polymerase enzyme. PCR-based techniques where random primers are used include random amplified polymorphic DNA (RAPD), arbitrarily primed PCR (AP– PCR) and DNA amplification fingerprinting (DAF). Inter simple sequence repeats (ISSRs) polymorphism is a specific primer-based polymorphism detection system, where a terminally anchored primer specific to a particular simple sequence repeat (SSR) is used to amplify the DNA between two opposed SSRs of the same type. Polymorphism occurs whenever one genome is missing in one of the SSRs or has a deletion or insertion that modifies the distance between the repeats. A recent approach known as amplified fragment length polymorphism (AFLP) is a technique that is based on the detection of genomic restriction fragments by PCR amplification. Adaptors are ligated to the ends of restriction fragments followed by amplification with adaptor-homologous primers.
AFLP has the capacity to detect thousands of independent loci and can be used for DNAs of any origin or complexity.
DNA sequencing can also be used as a definitive means for identifying species. Variations due to transversion, insertion or deletion can be assessed directly and information on a defined locus can be obtained. Genetic variation occurs extensively at the single nucleotide level. Direct sequencing can efficiently identify such single nucleotide polymorphisms that usually depend on how closely related are the organisms being compared. Other sequencing- based strategies include analysis of the variable internal transcribed spacer (ITS) sequences of ribosomal DNA (rDNA). The ITS region of 18s–26s rDNA has proved to be a useful sequence for phylogenetic studies in many angiosperm families. The level of ITS sequence variation suitable for phylogenetic analysis is found at various taxonomic levels within families, depending on the linkage. A number of researchers have also sequenced other regions of DNA such as trnK of chloroplast and spacer region of 5s rDNA as diagnostic tools for authentication purpose.
Few techniques in molecular biology have received so much attention and popular acceptance as PCR. Invented by Kary Mullis in 1983, PCR is a method used to generate billions of copies of genomic DNA within a very short time. This amplification is useful in criminal cases where there are miniscule amounts of DNA available. Today PCR finds application in almost all aspects of biomedicine. PCR has been used for the detection of many pathogenic organisms, from bacteria to viruses.
Figure1. The Polymerase Chain Reaction is used to amplify a sample of DNA.
Techniques used in DNA Fingerprinting
1. Microsatellites are simple sequence repeats (SSRs), 1 to 6 nucleotides in length, which show a high degree of polymorphism. Specific microsatellites can be isolated using hybridized probes followed by their sequencing. Like any DNA fragment, SSRs can be detected by specific dyes or by radiolabelling using gel electrophoresis. The advantage of using SSRs as molecular markers is the extent of polymorphism shown, which enables the detection of differences at multiple loci between strains .Coupled with chemical and morphological data, we can identify the plant species or strain of interest. The main advantage of using SSRs for fingerprinting is that small amounts of DNA are required compared to the restriction fragment length polymorphisms (RFLP) method. This is due to the large amounts of SSRs present in any genome. Further, assays involving SSRs are more robust than random amplified polymorphic DNA (RAPDs), making them up to seven times more efficient. A drawback to using SSRs is the need to develop separate SSR primer sets for each species. The latest research suggests that SSRs will be involved in new methods of detection of alterations of specific sequences in the DNA.
2. Restriction fragment length polymorphisms are unequal lengths of DNA fragments obtained by cutting Variable Number of Tandem Repeat (VNTRs) sequences up to 30 sequences long with restriction enzymes at specific sites. VNTRs vary between plant species, as do the number and location of restriction enzyme-recognition sites. On an agarose gel, RFLPs can be visualized using radiolabeled complementary DNA sequences. There is no need for PCR amplification of DNA in this method. A routine southern blot experiment is used instead. Normally, RFLPs are used to identify the origins of a particular plant species, setting the stage for mapping its evolution. There are some problems with the RFLP method of DNA fingerprinting. First, the results do not specifically indicate the chance of a match between two organisms. Secondly, the process involves a lot of money and labor, which not many laboratories can afford. Finally, unlike the microsatellites, a few loci in the assay must suffice.
Figure2. RFLP is one of the DNA fingerprinting techniques that is used to determine plant strain and purity in nutraceutical and herb production.
3. Amplified fragment length polymorphism (AFLP) is a PCR-based derivative method of RFLP in which sequences are selectively amplified using primers. It is a reliable and efficient method of detecting molecular markers. DNA is cut with two restriction enzymes to generate specific sequences, which are then amplified suitably. The mere addition or deletion of bases at the 3′ end determines the selectivity and complexity of the amplification 4. By using AFLP, it is possible to evaluate more loci than with RFLP or RAPD. AFLP is also capable of determining a large number of polymorphisms. Similar to SSRs, AFLP-based assays are cost-effective and can be automated.
4. Random amplified polymorphic DNA is one of the most commonly used primary assays for screening the differences in DNA sequences of two species of plants. RAPD consists of fishing for the sequence using random amplification. Here, plant genomic DNA is cut and amplified using short single primers at low annealing temperatures, resulting in amplification at multiple loci. By running a 2-dimensional electrophoresis gel, it is possible to determine the change in sequence pattern by superimposing the 2 gels. Once the band of interest is identified, the gel is cut, and the DNA is isolated and sequenced. Using this target, DNA from other cultivars can be assessed using other techniques such as AFLP or SSRs. It is also more cost effective than RFLPs. RAPDs lack specificity, however, due to low annealing temperatures and easier reaction conditions.
5. Other Methods include the use of single nucleotide polymorphs (SNPs) DNA amplification fingerprinting (DAF) and their offshoots. Although these techniques vary slightly from each other, they operate on the same principle.
Used for the betterment of drug-yield by tissue culturing. DNA of interest can be stored as germplasm, which is then used for future cultivation. In addition, germplasm can be used for the conservation of selected plant species, which are endangered such as Rauwolfia serpentina (Snake Root).
2. Legal Protection
Because of the uniqueness of DNA fingerprint data, the technique can be used to legally protect new varieties of plants or animals, whether they were developed by genetic engineering, tissue culture, or traditional methods. Using DNA fingerprints to identify and protect commercial varieties of crops or livestock is a relatively new application of DNA fingerprint technology.
3. Authentication of medicinal plants
Dried fruit samples of Lycium barbarum were differentiated from its related species using RAPD markers. The RAPD technique has also been used for determining the components of a Chinese herbal prescription, yu-pingfeng san. In this study the presence of three herbs (Astragalus membanaceus (Fisch.) Bge, Ledebouriella seseloides Wolff and Atractylodes macrocephala Koidz) in the formulation have been detected using a single RAPD primer. Three RAPD primers have been identified that could successfully discriminate between three species of Atractylodes, from Chinese formulation purchased from local markets. In another study, three random primers were used to reveal the genetic variability of Astragalus medicine materials sold in Taiwan market. SSCP analysis was also conducted on PCR products from the ITS-1 region of rDNA in order to differentiate the two Astragalus species. Primers have been designed for hybridization with the hypervariable ends of microsatellite loci that could reveal DNA-polymorphism among five Eucalyptus species. DAF has been used to identify the Chinese traditional medicine, Magnoliae officinalis, its counterfeits and substitutes. An RAPD primer that is selective for an elite strain Aizu K-111 of Panax ginseng, including its cultured tissues has been identified. RAPD and PCR– RFLP analysis have been used for authentication of P. ginseng among ginseng populations. Some researchers have used a new approach called Direct Amplification of Length Polymorphism (DALP) for authentication of Panax ginseng and Panax quinquefoliu. Authentication of medicinal Dendrobium species by the internal transcribed spacer of rDNA has been done successfully. A DNA microarray for detecting processed medicinal Dendrobium species (Herba dendrobii) was constructed by incorporating the ITS1-5.8s- ITS2 sequences of Dendrobium species on a glass slide. The established microarray could detect the presence of D. nobile in a Chinese medicinal formulation containing nine herbal components. Molecular authentication of Atractylodes-derived crude drugs (Jutsu) was done with the help of PCR–RFLP and direct sequencing of chloroplast trnK. Two regions (Region1 and Region2) inside the chloroplast trnK were selected as molecular markers for identification and discrimination of Atractylodes rhizome (Byaku-jutsu) and Atractylodes Lancea rhizome (So-jutsu). Based on polymorphism in the restiction site for Hinf1 in Region1 fragment (260 bp), it was possible to discriminate between the two species. By direct sequencing of Region 2 (436 bp) and comparison of the nucleotide sequence datasets, we could not only discriminate Byaku-jutsu and Sojutsu, but also identify the original plant species of each crude drug specimen.
Detection of adulteration/substitution
RAPD technique was adopted to identify eight types of dried Coptis rhizomes and one type of Picrorrhiza rhizome, a substitute for the former in the Chinese herbal market80. P. ginseng is often substituted by P. quinquefolius (American ginseng). Sequence characterized amplified region (SCAR), AP–PCR, RAPD and RFLP have been successfully applied for differentiation of these plants and to detect substitution by other closely related species81–83. Characterization of Echinacea species and detection of possible adulterations have been done using RAPD technique. DNA fingerprinting and polymorphism in the Chinese drug ‘Ku-Di-Dan’ (herba elephantopi) and its substitutes were studied using AP–PCR and RAPD. The results were used for authentication of ‘Ku- Di-Dan’ and its substitutes. DNA fingerprinting of Taraxacum mongolicum (herbal taraxaci) and its adulterants of six species of Compositae was demonstrated using AP–PCR and RAPD. Bulb of Fritillaria cirrhosa, an official drug of Chinese Pharmacopoeia (1995), is commonly used as an antitussive and expectorant. It has often been adulterated with similar bulbs of other related species. Specific DNA-based primers have been designed for authentication of F. cirrhosa at the genomic level. A molecular marker that is specific to medicinal rhubarb- based on chloroplast trnL/trnF sequence which is absent in its adulterants has been identified. DNA sequence analysis of rDNA ITS and PCR–RFLP were explored for their application in differentiating four medicinal Codonopsis species from their related adulterants, Campanumoea javania and Platycodon grandiflorus. The technique allowed effective and reliable differentiation of Codonopsis from the adulterants.
Fingerprinting of DNA is dictated by several factors; sequence or restriction site data, taxonomic level of study, the level at which the study is being done (species, genera, etc.), robustness and reproducibility of the method, effectiveness in terms of cost and time, and availability of DNA.
Although DNA analysis is currently considered to be cutting-edge technology, it has certain limitations due to which its use has been limited to academia. In order to establish a marker for identification of a particular species, DNA analysis of closely related species and/or varieties and common botanical contaminants and adulterants is necessary, which is a costly and time-consuming process. Isolation of good-quality DNA suitable for analysis from semi-processed or processed botanicals is also a challenge.
STANDARDISATION AND EVALUATION OF HERBAL DRUG FORMULATIONS
1Sunita Panchawat, 1Kamal Singh Rathore, 2Dr.S.S.Sisodia, 3Dr. R.K.Nema
1BN PG Girls College of Pharmacy, Udaipur-Raj.INDIA 313002
2BN PG College of Pharmacy, Udaipur
3Rishiraj College of Pharmacy, Udaipur
Mobile: +919887111211(Sunita P.); +919828325713(Kamal)
Herbal Drug Standardization and Evaluation:- In recent years, there has been great demand for plant derived products in developed countries. These products are increasingly being sought out as medicinal products, nutraceuticals and cosmetics. (1) There are around 6000 herbal manufacturers in India. More than 4000 units are producing Ayurveda medicines. Due to lack of infrastructures, skilled manpower reliable methods and stringent regulatory laws most of these manufacturers produce their product on very tentative basis. (2)
In order to have a good coordination between the quality of raw materials, in process materials and the final products, it has become essential to develop reliable, specific and sensitive quality control methods using a combination of classical and modern instrumental method of analysis. Standardization is an essential measurement for ensuring the quality control of the herbal drugs. (3) “Standardization” expression is used to describe all measures, which are taken during the manufacturing process and quality control leading to a reproducible quality. It also encompasses the entire field of study from birth of a plant to its clinical application. It also means adjusting the herbal drug preparation to a defined content of a constituent or a group of substances with known therapeutic activity respectively by adding excipients or by mixing herbal drugs or herbal drug preparations.(4) “Evaluation” of a drug means confirmation of its identity and determination of its quality and purity and detection of its nature of adulteration.(5)
Standardization of herbal drugs is not an easy task as numerous factors influence the bio efficacy and reproducible therapeutic effect. In order to obtain quality oriented herbal products, care should be taken right from the proper identification of plants, season and area of collection and their extraction and purification process and rationalizing the combination in case of polyherbal drugs.(3)
The Standardization of crude drug materials includes the following steps:-
Authentication: - Each and every step has to be authenticated.
a) Stage of collection.
b) Parts of the plant collected.
c) Regional status.
d) Botanical identity like phytomorphology, microscopical and histological analysis (characteristic of cell walls, cell contents, starch grains, calcium oxalate crystals, trichomes, fibers, vessels etc).(6) Various histological parameter studies are:-
Leaf constant: - Palisade ratio, Vein islet number, Vein termination, Stomatal number, and Stomatal index.
Ash values and extractive values.
Moisture content determination.
Chrometographic and spectroscopic evaluation.
Heavy metal determination.
The herbal formulation in general can be standardize schematically as to formulate the medicament using raw materials collected from different localities and a comparative chemical efficacy of different batches of formulation are to be observed. The preparations with better clinical efficacy are to be selected. After all the routine physical, chemical and pharmacological parameters are to be checked for all the batches to select the final finished product and to validate the whole manufacturing process. (6)
The stability parameters for the herbal formulations which include physical, chemical and microbiological parameters are as follow:
Physical parameters include color, odor, appearance, clarity, viscosity, moisture content, pH, disintegration time, friability, hardness, flow ability, flocculation, sedimentation, settling rate and ash values.
Chemical parameters include limit tests, chemical tests, chemical assays etc.
Chromatographic analysis of herbals can be done using TLC, HPLC, HPTLC, GC, UV, GC-MS, fluorimetry etc.
Microbiological parameters include total viable content, total mold count, total enterobacterial and their count. Limiters can be utilized as a quantitative or semi quantitative tool to ascertain and control the amount of impurities like the reagents used during abstraction of various herbs, impurities coming directly from the manufacturing vessels and from the solvents etc.
GUIDELINES FOR HERBAL DRUG STANDARDISATION
The subject of herbal drug standardization is massively wide and deep. The guidelines set by WHO can be summarized as follows:-
Reference to the identity of the drug. Botanical evaluation- sensory characters, foreign organic matter, microscopical, histological, histochemical evaluation, quantitative measurements etc.
Reference to the physicochemical character of the drug. Physical and chemical identity, chromatographic fingerprints, ash values, extractive values, moisture content, volatile oil and alkaloidal assays, quantitative estimation protocols etc.
Reference to the pharmacological parameters, biological activity profiles, bitterness values, hemolytic index, astringency, swelling factor, foaming index etc.
Toxicity details- pesticide residues, heavy metals, microbial contamination like total viable count, pathogens like E.coli, Salmonalla, P.aeroginosa, S. aureus, Enterobacteria etc.
Modern herbal ayurvedic monographs
In the modern herbal ayurvedic monographs the standardization parameters are discussed in a comprehensive way. According to the modern ayurvedic monograph the quality control protocols include the following:
The synonyms, publication related to the plant, constituents present, analytical methods.
Descriptive evaluation: Description of the drug, phytomorphological, microscopical, organoleptic evaluation, foreign matter etc.
WHO GUIDELINES MONOGRAPH TITLE (7)
Botanical: - Sensory evaluation, Foreign matter, Microscopy measurement.
Physicochemical TLC: - Ash, Extractable matter, Water content and volatile matter, Volatile oils.
Pharmacological: - Bitterness value, Haemolytic activity, Astringency, Sterling index, Foaming index.
Toxicological: - Pesticide residue, Arsenic, Metals.
Microbial contamination: - Total viable count, Pathogens, Aflatoxins, Radioactive contamination.
STANDARDIZATION OF HERBAL DRUG/PRODUCTS
Commercial production of herbal medicines and their trade are fast growing sector of industry today, due t6o increasing demand of medicinal plants; the supply line is adversely affected leading to the adulteration and substitution for genuine drugs.
1. Fluorescence quenching:- When a plant extract is spotted on a fluorescent silica gel layer and exposed to UV light, it appears as spot on a fluorescent background, thus causing quenching and is directly proportional to concentration of the extract. Silica gel GF plate was used as an adsorbent for fluorescence quenching. Solvents taken- hexane toluene, ether, ethyl acetate, butanol, methanol and water.(8)
2. Use of fingerprinting and marker compounds for identification and standardization of botanical drugs:- Chemical and chromatographic techniques may be used to aid in identification of an herbal material or extract. Chromatographic technique such as HPLC, TLC, GC and capillary electrophoresis and spectroscopic methods such as IR, NMR, and UV-may also be used for fingerprinting. DNA fingerprinting has been widely used in many species, e.g. DNA fingerprinting of Panax species and their adulterants.(9) Marker compounds may be used to help identify herbal materials, set specifications for raw materials, standardize botanical preparations during all aspects of manufacturing processes and obtain stability profiles.(10)
3. Densitometric thin layer chromatographic determination of aescin in an herbal medicinal product containing Asculum and Vitis dry extract:- A TLC method is developed to analyze the total saponin content, also referred to as the aescin content, in an herbal medicinal product containing two dry extract in capsules. After a purification step using C(18) solid phase extraction, the samples are analyzed on a silica gel HPTLC plate with the upper layer of a mixture of acetic acid/water/butanol(10/40/50v/v/v) as the mobile phase. Spots are visualized by spraying with anisaldehyde reagent and heating the plate for 5-10 min.(100-105oc) and measured at a wavelength of 535 nm.(11)
4. Determination of stigmasterol, beta-sitosterol and stigmastanol in oral dosage forms using HPLC with evaporative light scattering detection: - A validated and repeatable HPLC method with online evaporative light scattering was developed for the analysis of two sterols, stegmasterol, beta-sitosterol and a stanol found to be common in many herbal formulations and health care supplements. This method was used to assay commercially available products formulated as oral dosage forms purported to contain African potato and associated sterols and stanol. (12)
5. Elemental analysis of herbal preparations for traditional medicines by neutron activation analysis with the kO standardization method: - Medicinal herb preparations prescribed for specific treatment purposes were purchased from markets and were analysed by instrumental neutron activation analysis with kO standardization. 500-700 mg of each sample was palletized under a pressure of six tones and irradiated together with monitors for alpha and neutron flux ratio determination for about 6h in a thermal flux of 2.29 x 10(12) n/cm2/s.(13)
6. Liquid chromatography UV-determination and liquid chromatography-atmospheric pressure chemical ionization mass spectrometric characterization of sitosterol and stigmasterol in soyabean oil:- A narrow bore HPLC-UV method was developed for the analysis of two of the more abundant naturally occurring phytosterols in vegetable oils: sitosterol and stigmasterol. The method enabled detection of the compounds at a concentration of 0.42 µ/ml and quantization at concentration of 0.52 and 0.54 µ/ml for sitosterol and stigmasterol, respectively.(14)
7. Simultaneous determination of cinnamaldehyde, eugenol and paeonol in traditional Chinese medicinal preparations by capillary GC-FID: - A capillary GC method was established for simultaneous determination of cinnamaldehyde(CNMD), eugenol(EL) and paeonol(PL) in two traditional Chinese herbal medicinal preparations, Weitongding tablet (WTDT) and Guifu Dihuang pill (GDHP). The assays were based on a programmed temperature GC in a 30 m x 0.53 mm capillary column with nitrogen as carrier and FID detector. Good linearity were obtained over ranges of 0.45-0.452 mg/l CNMD, 0.31-0.625 mg/l EL, and 0.30-610 mg/l PL, respectively. (15)
8. HPTLC fingerprinting of marketed formulation containing Shankhpushpi: - These are the important Ayurveda formulations used for perinatal care of mother and child health. Standardization of churnas was carried out by organoleptic study, phytochemical analysis; qualitative organic and inorganic analysis, thin layer chromatography, UV- visible spectrophotometer and HPLC fingerprint studies. Qualitative organic analysis of both the churnas revealed the presence of alkaloids, steroids, phenols, tannins, glycosides, resins, saponins and flavonoids.(16)
EVALUATION OF HERBAL DRUG/PRODUCTS
1. Biological parameter (bioassay):- It is well established that the biological potency of the herbal constituents is due to not one but a mixture of bioactive plant constituents and the relative properties of a single bioactive compound can vary from batch to batch while the biological activity remains within the desirable limits. (1) Some of the examples are:_
a. Evaluation of adaptogenic activity profile of herbal preparation: - Adaptogens help the body to come up with stress and enhance general health and performance. AVM is an herbal formulation. Composition- Emblica officinalis, Withania somnifera, Asparagus racemosus, Ocimum sanctum, Tribulus terrestris and Piper longum. AVM shows significant antistress, immunomodulatory and anabolic activities in different animal models there by proving a promising adaptogen. (17)
b. Evaluation of antioxidant activity of herbal products: - A new test method for measuring the antioxidant power of herbal products, based on solid phase spectrophptometry using tetrabenzo-b, f, j, n, l, 5, 9, 13- tetraazacy- clohexadecin- Cu (II) complex immobilized on silica gel is proposed. The method represents an alternative to the mostly used scavenging capacity assays. The method was approved in the analysis of the most popular herbal beverages and drugs Echinacea determined spectrophotometrically.(18)
c. Evaluation of microbial contamination reduction on plants through technological process of decoction and spray dry: - The technological process of raw material has many stages, generally, adverse to microbial growth, but its complete elimination depends on the initial and work condition utilized. The aim of this work was to verify the microbial contamination, such as extractive solution (SE) and spray dried extract (PSA) with the purpose of evaluating the decrease of contamination after the decoction and the spray dry. The microbiological analysis of the products was performed by total plate count and MPN coliform. (19)
d. Evaluation of nitric oxide scavenging activity of selected medicinal plants used in inflammatory diseases: - Four traditional medicinal plants, namely Ventilago madraspatana Gaertn., Rubia cordifolia Linn., Lanatana camara Linn. And Morinda citrifolia Linn. Were selected for a study on the inhibition of nitric oxide (NO), a key mediator in the phenomenon of inflammation, signifying the presence of effective anti-inflammatory constituents therein. Plant samples were extracted with different solvents for evaluation of their inhibitory activity on NO produced in vitro from sodium nitroprusside, and in LPS- activated murine peritoneal macrophages, ex-vivo.(20)
e. The lipid peroxidation inhibitory activity:- The reaction mixture contained mice liver homogenate (0.2 ml, 10% w/v) in 0.15 KCl, KCl (0.1 ml, 150 µm), Tris buffer (0.4 ml, Ph 7.5) and various concentration of test extracts. In vitro lipid peroxidation was initiated by addition of Feso4.7H2O (0.1 ml, 10 µm). The reaction mixture was incubated at 37o for 1 h. After the incubation period, reaction was terminated by addition of thiobarbituric acid (TBA-2 ml, 0.8%) and by heating the contents for 15 min. for development of colored complex. The tubes were then centrifuged at 4000 rpm for 10 min. and cooled. The % inhibition of lipid peroxidation was determined by comparing the results of test compound with those of control not treated with extracts by monitoring the color intensity at 532 nm. Gallic acid was used as a positive control. (21)
2. Evaluation of marketed polyherbal antidiabetic formulatios using biomarker charantin: - Charantin is one of the phytoconstituents present in Momordica charantia. It is well known to possess antihyperglycaemia, anticholesterol, immunosuppressive, antiulcerogenic, antispermatogenic and androgenic activities. HPTLC method is fast, precise, sensitive and reproducible with good recoveries for standardization of polyherbal formulations. The recovery values of charantin were found to be about 98.89%. (2)
3. In vivo and in vitro evaluation of hair growth potential of Shoe flower: - The leaves and flowers of Hibiscus rosa-sinensis are used as promoters of hair growth and as an aid in healing of ulcers. Petroleum ether extract of leaves and flowers of the plant was evaluated for the potential growth in vivo and in vitro methods. In vivo, 1% extract of leaves and flowers in liquid was applied topically over the shaved skin of albino rats and monitored and assessed for 30 days. The length of hair and different cyclic phases of hair follicles, like anagen and telogen phases were determined at different time periods. In vitro, the hair follicles from albino rat neonates were isolated and cultured in DMEM supplemented with 0.01 mg/ml petroleum ether extract of leaves and flowers. It is concluded that the leaf extract, when compared to flower extract, exhibits more potency on hair growth. (22)
4. Clinical evaluation to assess the safety and efficacy of coded herbal medicine “Dysmo-off” versus allopathic medicine “Diclofenac sodium” for the treatment of primary dysmenorrhoea: - The clinical study on primary dysmenorrhoea to comparatively examine the coded herbal drug formulation “Dysmo-off” with authentic allopathic medicine “Diclofenac sodium”. A random controlled clinical trial was conducted. These evaluations were based on verbal rating scale so as to ascertain the rate of analgesic effects on dysmenorrhoeic pain. The patients were randomly allocated with the ratio of 1:2 for controlled treatment with (NSAIDS) (n=40) received Diclofenac sodium tablets twice daily for 4 days (50 mg one day prior to and three days after the menstruation), and test treatment with Dysmo-off (n=80) received powdered Dysmo-off twice daily for 4 days (5 g one day prior to and three days after the menstruation). Treatment lasted for 4 consecutive menstrual cycles. Haemoglobin, ESR and ultrasound were measured at baseline during study. All subjects were clinically studied.(23)
5. Thermographic evaluation: - In the present study, the authors used thermography to evaluate the effects of herbal formulations based on “Sho” scientifically. In the cases that were suitable for Keishibukuryogan, the so called Keishibukuryogan Sho, a significant skin temperature rise was observed in the upper half of the body after the intake of Keishibukuryogan. In a case that was suitable for Hochuekkito, a marked elevation of skin temperature spread through the upper trunk. It suggested that thermography is useful for an objective evaluation of Sho in Kampo medicines, and for identification of the action site of the herbal formulation.(24)
6. Biochemical evaluation: - Most of the herbal drugs are a mixture of a number of ingredients. Their cumulative effect increases the efficacy of the drug in curing the diseases. Muthu Marunthu is an herbal formulation comprising of eight various plant ingredients, and has been claimed to possess anticancer effect. It was observed that the growth rate in rats was normal and there was no change in blood parameters such as glucose, urea, proteins, cholesterol and also in the activities of pathophysiological enzymes such as lactate dehydrogenase (LDH), gluconate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), alkaline and acid phosphatase after Muthu Marunthu administration. The tumor weight was found to be reduced in methylcholanthrene induced fibrosarcoma rats after Muthu Marunthu treatment. (25)
7. Evaluation of Kutaj-Ghanavati for alkaloidal principles:- Kutaj-Ghanavati is a reputed Ayurvedic preparation used in dysentery and diarrhea. It contains water extract of Kurchi bark and fine powder of aconite roots. It was evaluated quantitatively and qualitatively employing TLC and titrimetric method. In TLC study no interference of Kurchi and Aconite alkaloids with one another in their respective solvent systems. The formulation was found to contain all alkaloids of Kurchi and Aconite. (26)
8. Organoleptic evaluation: - Organoleptic evaluation of food products plays an important role in judging the censoring acceptability or rejection of food items in the market. Effect of various treatments (blanching, pricking, and lye treatment), sugar concentration (50%, 60%, 70%) and storage on the color scores; flavor scores; texture scores of intermediate moisture apricots. The overall acceptability of the products was significantly higher in 70% sugar syrup but these scores decreased as the storage period advanced. (27)
CONCLUSION: - The subject of herbal drug standardization is massively wide and deep. There is so much to know and so much seemingly contradictory theories on the subject of herbal medicines and its relationship with human physiology and mental function.
For the purpose of research work on standardization of herbal formulations, a profound knowledge of the important herbs found in India and widely used in Ayurvedic formulation is of utmost importance.(6)
Even when the chemical composition of a plant extract is known, the pharmacologically active moiety may not be. Environment, climate, and growth conditions influence composition, as does the specific part of the plant and its maturity. Monographs detailing standardization of active ingredients would improve the marketplace. Even if an herbal product is standardized to, for example, 4% of a constituent, the remaining 96% of ingredients is not standardized and may affect the product’s solubility, bioavailability, stability, efficacy and toxicity. Just as controlled trials are necessary to establish safety and efficacy, manufacturing standards are required to ensure product quality.(28)
Now a days newer and advanced methods are available for the standardization of herbal drugs like fluorescence quenching, combination of chromatographic and spectrophotometric methods, biological assays, use of biomarkers in fingerprinting etc. Bioassay can play an important role in the standardization of herbal drugs and can also become an important quality control method as well as for proper stability testing of the product.(4)
India can emerge as the major country and play the lead role in the production of standardized, therapeutically effective ayurvedic formulation. India needs to explore the medicinally important plants. This can be achieved only if the herbal products are evaluated and analyzed using sophisticated modern techniques of standardization such as UV- visible, TLC, HPLC, HPTLC, GC-MS, spectrofluorimetric and other methods.(6)
Sagar Bhanu P.S., Zafar R., Panwar R., “Herbal drug standardization”, The Indian Pharmacist, vol. 4(35), May 2005, 2005, pp.19-22.
Patel P.M., Patel N.M., Goyal R.K., “Evaluation of marketed polyherbal antidiabetic formulations uses biomarker charantin”, The Pharma Review, vol.4 (22), June 2006, pp.113.
Patel P.M., Patel N.M., Goyal R.K., “Quality control of herbal products”, The Indian Pharmacist, vol.5(45), March 2006, pp.26-30.
Bhutani K.K., “Herbal medicines an enigma and challenge to science and directions for new initiatives”, Indian Journal of Natural Products, vol.19 (1), March 2003, pp.3-8.
Kokate C.K., Purohit A.P., Gokhale S.B., “Analytical pharmacognosy”, Pharmacognosy, 30th edition, Feb. 2005, pp.1,99.
Shrikumar S., Maheshwari U., Sughanti A., Ravi T.K., “WHO guidelines for herbal drug standardization”, 2006.
Ansari S.H., “Standardization of crude drugs”, Essentials of Pharmacognosy, Ist edition, 2005-06, pp.14, 581.
Gokhale S.B., Surana S.J., “Fluorescence quenching as a tool for identification and quality control of crude drugs”, Planta indica, vol 2 (3), July 2006, pp.47.
Shaw P.C., Pui-Hat Butt P., “Authentication of Panax species and their adulterants by random primed polymerase chain reaction”, Planta Medica, vol. 61, 1995, pp.466-469.
Lazarowych N.J., Pekos P., “Use of fingerprinting and marker compounds for identification and standardization of botanical drugs: Strategies for applying pharmaceutical HPLC analysis to herbal products”, Drug Information Journal, Vol.32, 1998, pp.497-512.
Apers S., Naessens T., Pieters L., Vlietinck A., “Densitometric thin-layer chromatographic determination of aescin in an herbal medicinal product containing Aesculus and Vitis dry extract”, Jr. of Chromatographic Analysis, vol.1112(1-2), April 2006, pp.165-170.
Nair V.D., Kanfer I., Hoogmartens J., “Determination of stigmasterol, beta-sitosterol and stigmastanol in oral dosage forms using HPLC with evaporative light scattering detection”, Journal of pharmaceutical and biomedical analysis, vol. 41(3), June 2006, pp. 731-737.
Sarmani S.B., Abugassa I., Hamzah A., Yahya M.D., “Elemental analysis of herbal preparations for traditional medicines by neutron activation analysis with the kO standardization method”, Biological trace element research, 1999, pp. 365-376.
Careri M., Elviri L., Mangia A., “Liquid chromatography-UV determination and liquid chromatography-atmospheric pressure chemical ionization mass spectrometric characterization of sitosterol and stigmasterol in soyabean oil”, Jr. of Chromatographic Analysis, vol. 935(1-2), Nov.2001, pp.249-257.
Yu B.S., Lai S.G., Tan QL, “Simultaneous determination of cinnamaldehyde, eugenol and paeonol in traditional Chinese medicinal preparations by capillary GC-FID”, Chemical and pharmaceutical bulletin, vol. 54(1), Jan 2006, pp. 114-116.
Santosh M.K., Shaila D., Sanjeeva Rao I., “Standardization study of dadimastaka and pushyannga churnas”, Asian Jr. of Chemistry, vol. 16(34), 2004, pp. 1735-1741.
Azamathulla Shaik, Hule Amolkumar, “Evaluation of adaptogenic activity profile of herbal preparation”, Indian Jr. of Experimental Biology, vol. 44, July 2006, pp.574-579.
Zaporozhets O.A., Lipkovska N.A., “A new test method for the evaluation of total antioxidant activity of herbal products”, Jr. of Agricultural and Food Chemistry, vol. 52(1), 2004, pp.21-25.
De Souza T.P., Zulian Lionzo M.I., “Evaluation of microbial contamination reduction on plants through technological process of decoction and spray dry”, Brazilian Jr. of Pharmacognosy, vol. 16(1), 2006, pp.94-98.
Basu S., Hazra B., “Evaluation of nitric oxide scavenging activity of selected medicinal plants used in inflammatory diseases”, Phytotherapy research, vol. 20(10), 2006, pp. 896-900.
Shinde A.D., Bhise S.B., “Evaluation of wound healing activity of herbal drug combination of Tridax Procumbens, Azadirachta indica, Curcuma longa and Apis mellifera”, Indian Drugs, vol. 41(6), June 2004, pp.376-378.
Adhiraj N., Ravikumar T., Shanmugasundaram N., “In vivo and in vitro evaluation of hair growth potential of Shoe flower”, Jr. of ethanopharmacology, vol. 88(2-3), 2003, pp. 235-239.
Nazar H., Usmanghani K., “Clinical evaluation toi assess the safety and efficacy of coded herbal medicine “Dysmo-off” versus allopathic medicine “Diclofenac sodium” for the treatment of primary dysmenorrhoea”, Jr. Herb Pharmacotherapy, vol. 6(1), 2006, pp.21-39.
Inokawa M., Iguchi K., Kohda H., “Thermographic evaluation of the efficacy of Kampo medicine”, Hiroshima Jr. Med Sci., vol. 55(1), March 2006, pp.1-8.
Palani V., Senthilkumaran R.K., “Biochemical evaluation in antitumour effect of Muthu Marunthu on experimental fibrosarcoma in rats”, Jr. of Ethanopharmacology, vol. 65(3), 1999, pp.257-265.
Bhavasar G.C., Pundarikakshudu K., “Evaluation of Kutaj-Ghanvati for alkaloidal principles”, Indian Jr. Natural Products, vol. 20(1), 2003, pp.33.
Sharma H.R., Verma P., “Organoleptic and chemical evaluation of osmotically processed Apricot wholes and halves”, Natural Product Radiance, vol.5 (3), Sep-Oct 2006, pp.350-356.
Boullata I.J., Nace M.A., “Safety issue with herbal medicine”, Pharmacotherapy, vol. 20(3), 2000, pp.257-269.
Retrieved from "http://www.articlesbase.com/alternative-medicine-articles/standardisation-and-evaluation-of-herbal-drug-formulations-1317004.html"
(ArticlesBase SC #1317004)
Read more: http://www.articlesbase.com/alternative-medicine-articles/standardisation-and-evaluation-of-herbal-drug-formulations-1317004.html#ixzz1745X6OKV
Under Creative Commons License: Attribution
SlideShare. Present yourself.
9 months ago
Login to your email and click the link to download the file directly.
Validation messages. Success message. Fail message.
Check your bulk/spam folders if you can't find our mail.
Select Email to import contacts
Yahoo! Mail Gmail Google Mail Hotmail MSN AOL
Are you a SlideShare user? Login to share with your contacts.
Email sent. Want to share with more friends?
Like this presentation?
CHEMICAL AND BIOLOGICAL/TOXICOLOGICAL STANDARDIZATION OF HERBAL
MCOPS , MANIPAL UNIVERSITY
Standardization of drug means confirmation of its identity and determination of its quality and purity and detection of nature of adulterant by various parameters like morphological, microscopical, physical, chemical and biological observations.
techniques involved in standardization of crude drugs
STANDARDIZATIONOF HERBAL DRUGS:
Moisture content and volatile matter
Viscosity of a liquid is constant at a given temperature and is an index of its composition. Hence, it can be used as a means of standardizing liquid drugs.
In case of pure photochemical, melting points are very sharp and constant.
The crude drugs from plant or animal origin, containing the mixed chemicals, are described with certain range of melting point.
Their purity can be ascertained by determining their melting points in that range
for E.g. Colophony- 75-80˚c
Cocoa butter- 30-33˚c
The presence of adulterant could be indicated by solubility studies
E.g.pureAsafoetida is soluble in carbon disulphide
MOISTURE CONTENT AND VOLATILE MATTER
The moisture content of the drug should be minimized in order to prevent decomposition of crude drug either due to chemical change or microbial contamination.
The moisture content is determined by heating a drug at 105˚c in an oven to a constant weight.
For the drugs containing volatile constituents, toulene distillation method is used
E.g. – Aloe should have moisture content not more than 10% w/w
Optically active compounds have the property of rotating the plane of polarized light.Thisproperty is known as optical rotation.
Normally, the optical rotation is determined at 25˚c using sodium lamp as the source of light.
E.g. castor oil has optical rotation from +3.5˚to +6˚
When a ray of light passes from one medium to another of different density, then the ratio of velocity of light in vaccum to its velocity in substance is termed as refractive index of second medium.
It is constant for a pure drug and varied with wavelength of incident light, temperature and pressure
E.g. Castor oil has refractive index 1.4758-1.527
VALUES AND EXTRACTIVES
The residue remaining after incineration is the ash content of drug
Total ash method is used to measure the total amount of material remaining after incineration
Acid insoluble ash is the residue obtained after boiling the total ash with dil. HCl and igniting the remaining insoluble matter.
Water soluble ash is the difference in weight between total ash and residue after treatment of total ash with water.
place 4 gms powdered material in a conical flask. Add water and weigh to obtain total weight.
Shake and allowed to stand for 1hr. attach the reflux condenser and boil for 1hr.
Readjust to the original weight with solvent. Shake and filter.
Transfer the filter to a flat bottomed disk and evaporate to dryness on a water bath.
Dry at 105˚ c for 6hrs, cool and weigh immediately.
Calculate the content of extractable matter in mg per g of air dried material.
Place the powdered material in a conical flask.
Macerate with 100ml of solvent specified for 6hrs, shake then allowed to stand for 18hrs.
Filter and transfer the filtrate to flat bottomed disk and evaporate to dryness on a water bath.
Dry at 105̊ c for 6hrs, cool and weigh immediately.
Calculated the content of extractable matter in mg per g of air dried material.
Medicinal plants having strong bitter taste are therapeutically used as appetizing agents
The bitterness is determined by comparing the threshold bitter concentration of an extract material with that of quinine hydrochloride
The bitterness value is expressed as units equivalent to the bitterness of a solution containing 1gm of quinine hydrochloride in 2000ml.
0.1gm of quinine hydrochloride is dissolved in 100ml drinking water and the stock solution is prepared. Then it is diluted and tested and compared with drug.
Bitterness value in unit per gm = 2000*c
Where, A = concentration of stock solution
B = volume of test solution in tube with threshold bitter concentration
C = quantity of quinine hydrochloride in the tube with threshold bitter concentration
Haemolyticactivity of plant material is determined by comparison with that of reference material, Saponin R, having haemolytic activity of 1000units/g.
Method of preparation of standard:
Fill a glass stopper flask to 1/10 of its volume with sodium citrate. Ass sufficient volume of blood freshly collected from healthy ox and shake, this can be stored for about 8 days at 2-4̊ c. place 1ml of citrated blood in a volumetric flask with phosphate buffer pH 7.4.
Haemolytic activity = 1000* a/b
Where, 1000 = defined haemolytic activity of Saponinstandard
a = quantityofsaponin standard that produce total haemolysis(g)
b = quantity of plant material that produce total haemolysis (g)
The swelling index is the volume in ml taken up by the swelling of 1gm of plant material under specified conditions.Its determination is based on addition of water or a swelling agent as described in test procedure.
The foaming ability of an aqueous decoction of plant material and their extracts is measured in terms of foaming index.
WATER AND VOLATILE MATTER:
Azeotropic method is used to directly measure the water present in a material.
Loss on drying
In order to measure volatile matter, plant is diluted with water and distillate is collected in a graduated tube. The aqueous portion separates and returns to distillation flask. A solvent of low mass density with a suitable boiling point may be added to measuring tube to easily separate the volatile oil.
METHODS OF STANDARDIZATION OF HERBAL DRUGS:
It comprises of different chemical tests and assays. The isolation, purification and identification of active constituents are chemical methods of evaluation.
Quantitative chemical tests shuch as acid vale, saponification value etc., are also coveredunder this technique. Qualitative chemical tests are used in detection of adulteration.
The chemical evaluation also covers phytochemical screening carried out for establishing chemical profile of a drug.
Detection of alkaloids
Detection of carbohydrates and glycosides
Detection of phytosterols
Detection of fixed oils and fats
Detection of saponins
Detection of phenolic compounds and tannins
Detection of protein and free amino acids
Detection of gums and mucilage
Detection of volatile oils
Drugs which cannot be assayed by chemical, or physical means are evaluated by biological methods
INDICATION FOR BIOLOGICAL EVALUATION
This is true for the substances having an
When quantity is too small.
No specific chemical test is available
When the action of drug is due to a mixture of substance
Purification of drug is not possible
When the estimation of crude drug or its preparation is done by means of its effect on living organism like bacteria, fungi, or animal tissue or entire animal it is known as BIOASSAY.
TYPES OF BIOASSAY
QUANTAL:-It is all or none phenomenon
GRADED:-Based on observation that there is a proportionate increase in the observed response with increase in concentration or dose.
bioassay can be performed by using any of the following
Multiple point bioassay
Determination of pesticides.
Determination of arsenic and heavy metals
Determination radioactive contamination
Determination of aflatoxins.
ARL = ADI*E*60
ADI=maximum acceptable daily intake of pesticides (mg/kg of body weight)
E= extraction factor, which determines the transition rate of the pesticides from the plant material into the dosage form
MDI=Mean Daily Intake of medicinal palnt products
60 in numerator=adult body weight
100 in denominator=consumption factor
of arsenic and heavy metals
Arsenic and heavy metals are even in trace amounts but they are dangerous removed from herbal drugs.
Amount is estimated by matching the depth of colour with of standard stain
Add water nitric acid and then sulphuric acid
Material is destroyed
No further darkening with heating
Clear solution with sulphur trioxide vapors, cool and add ammonium oxalate