3.2 Stability-Testing-of-Phytopharmaceuticals.pptx

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1. I/II M. PHARM I SEMESTER ADVANCED PHARMACEUTICAL ANALYSIS UNIT – 03 STABILITY TESTING OF PHYTOPHARMACEUTICALS 1

2. S YLLABUS 2

3. CONTENTS Introduction Advantages and Disadvantages of herbal drugs Standardization of herbal drugs Stability testing of herbal products Recommended protocols for standardization of herbal drugs Challenges in Stability testing of herbal medicinal product Regulatory requirements Role of markers in determining the stability of herbal drugs Analytical methods to determine stability of herbal products HPTLC/HPLC finger printing, interactions and complexity. Herbal -Drug interctions 3

4. INTRODUCTION Phytopharmaceuticals or Herbal drugs referred as plants materials or herbalism, involves the use of whole plants or parts of plants, to treat injuries or illnesses. Herbal drugs are the use of therapeutic herbs to prevent and treat diseases and ailments or to support health and healing. These are drugs or preparations made from a plant or plants and used for any of such purposes. Herbal drugs are the oldest form of health care known to mankind. 4

5. Nowadays, due to side effects of allopathic drugs, herbal drugs are prefered . Herbal products have reached extensive adequacy as beneficial agents like antimicrobial, antidiabetic, antifertility, antiageing , antiarthritic, sedative, antidepressant, antianxiety, antispasmodic, analgesic, anti-inflammatory, anti-HIV, vasodilatory, hepatoprotective, treatment of cirrhosis, asthma, acne, impotence, menopause, migraine, gall stones, chronic fatigue, Alzheimer's disease, and memory enhancing activities. 5

6. These drugs have survived real world testing and thousands of years of human testing. Some drugs have been discontinued due to their toxicity, while others have been modified or combined with additional herbs to counterbalance side effects. There are many herbal products offered that assert to treat the symptoms of a broad range of problems, from depression to cold and flu. 6

7. ADVANTAGES OF HERBAL DRUGS Low/Minimum cost Potency and efficiency Enhanced tolerance More protection Fewer side-effects Complete accessibility Recyclable 7

8. DISADVANTAGES OF HERBAL DRUGS Not able to cure rapid sickness and accidents Risk with self-dosing Complexity in standardizations 8

9. STANDARDIZATION OF HERBAL DRUGS Standardized herbal products of consistent quality and containing well-defined constituents are required for reliable clinical trials and to provide consistent beneficial therapeutic effects. Pharmacological properties of an herbal formulation depend on phytochemical constituents present therein. Development of authentic analytical methods which can reliably profile the phytochemical composition, including quantitative analyses of marker/bioactive compounds and other major constituents, is a major challenge to scientists. 9

10. Without consistent quality of a phytochemical mixture, a consistent pharmacological effect is not expected. Standardization is the first step for the establishment of a consistent biological activity, a consistent chemical profile, or simply a quality assurance program for production and manufacturing. 10

11. Therefore, the EU has defined three categories of herbal products: 1. Those containing constituents (single compounds or families of compounds) with known and experienced therapeutic activity that are deemed solely responsible for clinical efficacy. 2. Those containing chemically defined constituents possessing relevant pharmacological properties which are likely to contribute to the clinical efficacy. 3. Those in which no constituents have been identified as being responsible for the therapeutic activity. 11

12. Standardization means adjusting the herbal drug preparation to a defined content of a constituent or group of substances with known therapeutic activity. The European Medicines Agency (EMEA) makes the distinction between constituents with a known therapeutic activity which can be used to standardize a biological effect and marker compounds which allow standardization on a set amount of the chosen compound. 12

13. The EMEA defines marker compounds as chemically defined constituents of a herbal drug which are of interest for control purposes. Examples of markers are the valerenic acids in Valeriana officinalis L., ginkgolides and flavonoids in Ginkgo biloba L. and hypericin and hyperforin in Hypericum perfoliatum., independent of whether they have any therapeutic activity or not. 13

14. STABILITY TESTING OF HERBAL PRODUCTS Stability testing is necessary to ensure the product is of acceptable quality throughout its entire storage period. An important part of quality control of herbal products is the evaluation of the chemical stability of a finished product during the storage period. Stability testing of herbal products is a challenging task because the entire herb or herbal product is regarded as the active substance, regardless of whether constituents with defined therapeutic activity are known. 14

15. The objective of a stability testing is to provide evidence on how the quality of the herbal products varies with stability. Stability testing examines the quality and potency of drug at suitable time intervals under the influence of environmental factors such as temperature, light, oxygen, moisture, other ingredient or excipient in the dosage form, particle size of drug, microbial contamination, trace metal contamination, leaching from the container. Products are normally required to have shelf lifes that are measured in years. Accelerated stability studies are designed to increase the rate of chemical degradation or physical change of a drug substance, therefore, tests must also be conducted under conditions, which accelerate any changes occurring at ambient temperature and humidity. 15

16. Types of Stability Type Condition to be maintained Chemical Chemical integrity & labeled potency Physical Appearance & palatability , uniformity Microbiological Sterile Therapeutic Drug should remain potent Toxic Should not be toxic 16

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18. Storage conditions Stability study Storage conditions   Testing frequency (months)   Accelerated 40 ± 2ºC & 75 ± 5% RH   0,1,2,3 & 6   Intermediate   30 ± 2ºC & 65 ± 5% RH   0,3,6,9,12,18,24 & 36   Long term   25 ± 2ºC & 60 ± 5% RH   0,3,6,9,12,18,22,24,26,36,48 & 60   18

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20. RECOMMENDED PROTOCOLS FOR STANDARDIZATION OF HERBAL DRUGS In order to assure a consistent and acceptable quality herbal product, care should be taken right from the identification and authentication of herbal raw materials to the verification process of final product. It is desirable to establish a document database containing information on each approved medicinal herb or herbal medicine. 20

21. A central digital document database which is regularly updated and which contains this information with linkages to references in other databases like NAPRALERT should be established for easy access by all beneficiaries, producers, and stakeholders. The knowledge base for an herb or herbal medicine, promoted for wider use, should be strengthened and expanded so that there is a sound scientific basis for each use. 21

22. This would require the presentation of data for each herb, used in herbal medicine including: • plant identification, including cultivar with voucher specimen • plant, preferably voucher stored in a herbarium, for future reference • age of the herb (maturity/flowering plant, etc.) • location of cultivation, including altitude and longitude/latitude (GPS) • fertilizers/pesticides used, if any • time of harvest, including time after application of pesticides (if applicable) • storage conditions of the plant, before sale • drying process • certificate, confirming the above 22

23. Apart from details on each herb, details regarding the manufacture of the herbal medicine including the following data should be made available: • protocol or pharmacopoeia or method used for producing the medicine • plant or plants (for multi-plant herbal medicine) used • part of plant used in medicine • vehicle used for producing drug/medicine, e.g., alcohol or water (with composition if a mixed solvent); type of preservative used, if any, Where plants are purchased, documents maintained by the supplier regarding the herb should be made available by the manufacturer. 23

24. Prepare monographs for traditionally used herbal medicines in a suitable format. Legislate to ensure that manufacturers provide relevant data on herbs and manufacturing processes. Initiate programs to conserve biological resources. Work toward amending TRIPS to include protection of IPR contained in indigenous knowledge and to make the development of herbal medicines attractive to pharmaceutical companies. 24

25. Legislate to establish standards in herbal medicine manufacturing and ensure their implementation. Patents involving biological resources should be granted only if the source of material is specified and reference made to the material transfer agreement. As most herbal medicines are prepared from more than one plant material, it is imperative that documentation should be made both on single medicinal plants and on composite herbal preparations 25

26. Single plants • plant identification: family, genus, species (including cultivar, if any) with synonyms and older names where applicable; English (common) name(s); local name • herbarium voucher; specimen number and date with collector‘s name and identity • age of the herb (maturity/ flowering plant, etc.) • location of cultivation/collection including altitude and longitude/latitude (GPS) 26

27. • time of harvest including time after application of pesticides (if applicable) • storage conditions of the plant before sale • drying process • certificate confirming the above 27

28. Composite herbal medicine Apart from details on each herb as outlined for single plant, details regarding the manufacture of the composite herbal medicine, including the following data, should be made available: protocol or pharmacopoeia or method used for producing the medicine plant or plants (for multi-plant herbal medicine) used part of plant or plants used in the medicine vehicle used for producing the drug/medicine, e.g., alcohol or water (with composition if a mixed solvent); type of preservative used, if any, and amount excipients (if any) used, amount 28

29. • major constituents: carbohydrates, protein, fat, dietary fiber , inorganic material, binder, total energy value per mass (kcal/g or kJ/g) • suggested dose, number of times to be used, how many days to be used • probable side effects/precautions to be taken, contraindications, restrictions for children, pregnancy, nursing mothers, etc. • storage: at x °C, room temperature, away from heat, sun, etc. • stability; shelf life; best before date • cautionary note, if any 29

30. CHALLENGES IN STABILITY TESTING OF HERBAL MEDICINAL PRODUCT Evaluating the stability of HMPs presents a number of challenges when compared to chemically defined substances. In particular: 1. Active substances (herbal substances and/or herbal preparations) in HMPs consist of complex mixtures of constituents and in most cases the constituents responsible for the therapeutic effects are unknown. 2. The situation is further complicated when two or more herbal substances and/or herbal preparations are combined in a HMP. 30

31. In many cases where combinations of herbal substances and/or herbal preparations are present in HMPs, they have similar constituents and this gives rise to even more analytical challenges. 3. In addition, many herbal substances/herbal preparations are known to be unstable. 31

32. Taking into account these special features of HMPs, adequate quality concepts have been established. As part of a total control strategy for herbal substances, herbal preparations and HMPs, a set of test criteria including qualitative and quantitative parameters has been recognized as quality indicating. With regard to stability tests, chromatographic fingerprints as well as appropriate methods of assay via marker substances represent the fundamental part of this concept, laid down in shelf-life specifications. 32

33. Not withstanding the appropriateness of this approach, its realization is often associated with analytical problems and high costs. In summary, HMPs have a number of characteristics that clearly differentiate them from chemically defined medicinal products and therefore specific stability guidance needs to be established, which covers particular aspects that existing specific herbal guidelines and general guidelines on stability do not address. 33

34. WHO GUIDELINE – Assessment for modern herbal medicines Guideline available on following: Pharmaceutical assessment Crude plant material Plant preparations Finished product Stability assessment and shelf life Activity Evidence required to support indication 34

35. These guidelines describe.. Quality control of crude drugs material, plant preparations and finished products. Stability assessment and shelf life. Safety assessment documentation of safety based on experience or toxicological studies. Assessment of efficacy by medical information and biological activity evaluations. 35

36. Following new points are included: 1. Limits of pesticides Mainly pesticides like DDT, BHC, toxaphene and aldrin cause serious side effects in human beings if the crude drugs are mixed with these agents. 2. Limits of microbial contamination. 3. Radioactive contamination. 36

37. REGULATORY REQUIREMENTS The regulatory scenario regarding herbal preparations varies from country to country. Regulation in India Herbal drugs are regulated under the Drug and Cosmetic Act ( D and C) 1940 and Rules 1945 in India, where regulatory provisions for Ayurveda, Unani, Siddha medicine are clearly laid down. Department of AYUSH is the regulatory authority and mandate that any manufacture or marketing of herbal drugs have to be done after obtaining manufacturing license, as applicable. 37

38. The D and C Act extends the control over licensing, formulation composition, manufacture, labelling, packing, quality, and export. Schedule “T’’ of the act lays down the good manufacturing practice (GMP) requirements to be followed for the manufacture of herbal medicines. The official pharmacopoeias and formularies are available for the quality standards of the medicines. First schedule of the D and C Act has listed authorized texts, which have to be followed for licensing any herbal product under the two categories: ASU drugs and patent or proprietary medicines. 38

39. In India, traditional medicines are governed by the Drugs and Cosmetics Act of 1940 and the Drugs and Cosmetics Rules of 1945. They regulate the import, manufacture, distribution and sale of drugs and cosmetics. In 1959, the govt of India recognized the traditional Indian systems of medicine and amended the Drugs and Cosmetics Act to include drugs which are derived from traditional Indian medicine. No products derived from traditional systems may be manufactured without a license from the state Drug control Authorities. 39

40. Patent and proprietary medicines derived from the traditional systems must contain ingredients which are mentioned in the recognized books of the above systems, as specified in the Drugs and Cosmetics Act. The government is advised by a special committee and an advisory board for Ayurvedic, Siddha and Unani drugs. Pharmacopeia committees have been constituted to prepare pharmacopoeias for all these systems. 40

41. In Europe, for the marketing approval, the herbal preparations are classified in three categories as follows: Traditional medicinal use provisions (“traditional use”) accepted on the basis of sufficient safety data and plausible efficacy. Well-established medicinal use provisions (“well-established use”) demonstrated with the provision of scientific literature establishing that the active substances of the medicinal products have been in well-established medicinal use within the European Union for at least 10 years, with recognized efficacy and an acceptable level of safety a product can be classified under. 41

42. ROLE OF MARKERS IN DETERMINING THE STABILITY OF HERBAL DRUGS Markers are chemically known compounds, which may or may not have therapeutic effect, they are used to calculate the quantity of herbal medicinal ingredients in herbal medicinal products. It is important to isolate and structurally elucidate chemically defined substances in plants, drug and/ or drug preparations so that they can be used as markers that not only help to better understand the active principles of herbal drugs but also can enhance analytical quality control. 42

43. ANALYTICAL METHODS TO DETERMINE STABILITY OF HERBAL PRODUCTS The analysis of herbal preparations is mostly done by modern chromatographic or Spectroscopic methods like HPLC, gas chromatography (GC), TLC, quantitative determinations by UV Visible spectroscopy or combinations of these.  HPLC and GC methods can be used for identification and purity testing, as well as the detection of single compounds for assay, is possible during one analysis. LC and GC mass coupling are also the tools for determination. 43

44. Fingerprint analysis Fingerprint analysis approach is the most potent tool for quality control of herbal medicines because of its accuracy and reliability. Fingerprinting is a process that determines the concentrations of a set of characteristic chemical substances in an herb. Knowing the relative concentrations is a means of assuring the quality of herbal preparations. It can serve as a tool for identification, authentication and quality control of herbal drugs. 44

45. Based on the conception of phytoequivalence , the chromatographic fingerprinting and DNA fingerprints of herbal medicines could be utilized for addressing the problem of quality control of herbal medicines 45

46. Chromatographic fingerprinting: Chromatographic fingerprinting is the most powerful approach for the quality control of herbal medicines. Chromatographic fingerprint of Herbal Medicine is a chromatographic pattern produced from extract of some common chemical components which may be pharmacologically active or have some chemical characteristics. 46

47. Chromatographic fingerprinting can be carried out using techniques such as Thin layer chromatography (TLC), High performance thin layer chromatography (HPTLC), High performance liquid chromatography (HPLC), Gas chromatography (GC) and other hyphenated techniques. 47

48. High Performance Thin Layer Chromatography (HPTLC) HPTLC is the common fingerprinting method mainly used to analyze the compounds with low or moderate polarities. HPTLC technique is widely used in the pharmaceutical industry for quality control of herbs and health products, identification and detection of adulterants, substituents in the herbal products and also helps in the identification of pesticide contents and Mycotoxins. HPTLC method has several advantages which are as follows: 48

49. Advantages Several samples can be run simultaneously by use of a smaller quantity of mobile phase as compared to HPLC. Mobile phases of pH 8 and above can be used for HPTLC. Repeated detection (scanning) of the chromatogram with the same or different conditions. HPTLC has been investigated for simultaneous assay of several components in a multi-component formulation. 49

50. HPTLC technique was reported for simultaneous determination of betasitosterol -d glucoside and withaferin A in Ashwagandha formulations using silica gel GF60 as stationary phase and Chloroform: Methanol (8:2) as mobile phase. Retention factor was found to be 0.21 (peak 1) and 0.59 (peak 2), respectively 50

51. HPTLC technique is widely employed in pharmaceutical industry in process development, identification and detection of adulterants in herbal product and helps in identification of pesticide content, mycotoxins and in quality control of herbs and health foods 51

52. High Performance Liquid Chromatography (HPLC) The preparative and analytical HPLC has been widely used for analysis of herbal medicines because of its high separation capacity. It can be employed to analyze almost all constituents of herbal products provided that an optimized procedure is developed which involves optimization of mobile phase and stationary phase along with other chromatographic parameters. 52

53. There are basically two type of Preparative HPLC: Low pressure HPLC (typically under 5 bar) High pressure HPLC (pressure >20 bar). Reversed phase columns are the most popular columns used in the analytical separation of herbal medicines. 53

54. The objective of the works was to establish the best way to have appropriate fingerprints for all the main phytochemicals present in the samples. The relationship between the fingerprints was notified. For traditional quality control system, rosmarinic acid, curcumin, andrographolide and cinnamaldehyde are used as the marker substances to evaluate the extract of JD plant materials. 54

55. The contents of these compounds present in the sample were determined by linear regression analysis. To determine the linearity equations and linear scope for the analysts, a series of standard solutions at different concentrations were tested. All results indicated that the conditions for the fingerprint analysis were satisfactory. 55

56. HPLC Chromatogram of rosmarinic acid standard marker 56

57. HERBAL- DRUG INTERACTIONS: Herbs are often administered in combination with therapeutic drugs, raising the potential for herb-drug interactions. Cases have been published reporting enhanced anticoagulation and bleeding when patients on long-term warfarin therapy also took Salvia miltiorrhiza ( danshen ). Allium sativum (garlic) decreased the area under the plasma concentration-time curve (AUC) and maximum plasma concentration of saquinavir, but not ritonavir and paracetamol (acetaminophen), in volunteers. 57

58. Herb drugs + Allopathic drug = Some Reactions When herbal medicinal products and western drugs administered together may interact each other in body leading to kinetic and dynamic alterations. 2. Herbs are often administered in combination with therapeutic drugs, raising the potential of herb-drug interactions. 3. Herbs or Herbal drugs often taken with the Allopathic drugs with belief that it will have some Beneficial effect. 4. Most of the herbal drugs are taken because of- Availability, Economic consideration and its safety 58

59. Reason for herb-drug interacion 1.Clinician lack of adequate knowledge about Drug-herb Interaction 2. No quality control and assurance for the purity and safety. 3. No advance research in this field. 4. Blind believe or over believe in Ayurverdic medicine 5. Avoidance of patient history about drug sensitivity 6. Adulteration in herbal drug 59

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64. QUESTIONS Write about HPTLC finger printing technique in stability testing of phytopharmaceuticals? Write a short note on regulatory requirements of stability testing of phytopharmaceuticals? 64

65. REFERENCES 1. Drug stability by Cartensen 2. Pharmaceutical dosage forms by Aulton 3.Hand book of stability testing in pharmaceutical development(regulations, methodologies & best practices) edited by Kim Huynh 65

66. VIDEO LINKS https://youtu.be/2GY https://youtu.be/A_jxyxJUNJc https://youtu.be/dNueDQ0Fn9U 66