Welcome to Lavanya Ayurveda

1. Welcome to LavanyaAyurveda

2. World Humanity needs Integration of all Pathies because of merits and limitations of each pathy

3. Lavanya’s Vision • Our vision is to see a healthier world with the help of ancient Ayurvedic achievements and new age scientific technology. We aspire to blend the science of Ayurveda with modern medical research methodology to provide natural solution to human ailments.

4. Latest Technologies to diagnose at Physical Level. Modern Science treatment has Merit of Surgery, Antibiotics, Emergency and Symptomatic treatments on physical level. But at the same time it has its own limitations. But in Ayurveda the Diagnosis is made on Physical (Constitutional), Mental (Emotional) and Soul Level. Ayurveda has 2000 years old documentation but due to the lack of scientific evaluation Modern Science & Ayurveda • Modern • Ayurveda

5. Modern Science & Ayurveda Modern • It is far behind the modern system of medicine. Since past few years we are working on scientific parameters.

6. Quality analysis & standardization A. For AyurvedicBhasma classical BhasmaPariksha • Rekhapurna (Bhasma particles enters in to furrows of the finger. This indicates the fineness of the particles.) • Varitar (floats over the stable water surface. This test indicates the lightness of Bhasma) 3. Unam (Putting a grain over the floating particle) 4. Nirdhum (does not yield fumes over fire) 5. Nishchandra (absence of metallic lustre)

7. Quality analysis & standardization B. Elemental assay for Bhasma by AAS method at Biotech Park Lab. C. Raw Material & finished product are analyzed and compared with standard (API)TLC, Total Ash Acid Insoluble Ash, Alcohol and water soluble extractive, loss on drying at 105 ◦ C D. Isolated components for standardization of different samples (eg. Humic acid & Fluvic acid for Shilajeet by HPLC) wherever applicable.

8. Lavanya’s R & D Division

9. our aim • Researches on :- • Cancer: Second commonest cause of death. • AIDS:Dreaded disease of the decade. • Diseases Refractory to modern treatment e.g. Hepatitis, RA, Osteoporosis etc. • Evaluation of Ayurvedic drugs using Modern Parameters for their efficacy and safety. • Evaluation of Ayurvedic Diagnostic proceduresand to correlate them with modern parameters. • Modification of the dosage form by Nano Technology. • In-vitro Studies: Cell line, Cell cycle (Flow Cytometry) and current Tissue Culture technique. • Quality Analysis with Standard and to develop current method of Standardization of drugs which are not mentioned in API.

10. In-vitro cell line study

11. In-vitro Cyto Chemical & Flow-cytometry – Facility provided by CDRI, Lucknow • Cancer cell lines used were breast cancer (MCF7), cervical cancer (HELA), colon cancer (COLO-205) and prostate cancer (DU-145) • LAS02 inhibits proliferation by inducing retention of cells by 79.28% at 400 µg/ml of dose in G0/G1 phase along with apoptosis induced 43.51% at same dose significantly at in-vitro level.

12. Flow- cytometric responses with 400µg/ml dose of LAS02 % of Apoptosis & Retention of Cell Apoptosis Diploid G1 Diploid G2 Diploid S 400 µg/ml

13. Maximum Response in G0/G1 Phase of the Cell Cycle as seen by Flow-cytometry • (A) Cell cycle result for 24 hrs. in Colon Cancer Cell Line • (B) Cell cycle result for 48 hrs in Prostate Cancer Cell Line

14. MG63- osteosarcoma cell line

15. Expression of Proteins

16. Expression of Proteins

18. Expression of Proteins p53. • Effect on Expression of TSG,POG & anti ROS Parameters. • p53 • Bcl2 • Caspase • Survivin • NF Kappa B • LAS02 a Cancero-static compound enhanced the expression of • p53 in different type of malignancies. • p53 values significantly (4.76U/ml 35.94U/ml).

19. Effect on Hormones profiles

20. Chakras (Energy stores) Kundalinijagran modifies hormonal profile by LAS01- • 1. Antistress – A/ACTH/CARTICOIDS • Pineal Gland (3rd eye):- • Melatonin, Serotonin. • Other releasing hormones and • targeted hormones is under study with ELISA by using Kits. • Effect of selected Ayurvedic • drugs on hormonal profile such as • A. LAS-01 • B. Energy management

21. In Vitro USA study

22. Isolated CANCER Cell lines or naturally Cultured cancer tissues for screening anti cancer drug (USA).

23. Results & Conclusion • Live cell imagining and proliferation data demonstrate: • Most effective and time-depended anti-tumor activity was observed at 25mg/ml treatment dose of Las01 agent with MDA0132 and HT-29 Microtumors.

24. Isolation of DNA & Sequencing

25. Expression analysis of several proteins expressed during Cancer • The effect and change on the genetic and expression level has to be analyzed by studying the protein expressed by the genes related to cancer and modulation or mutation that bring these effects and changes can be analyzed so as to know the exact drug activity and pattern of action on various cancerous cells. The studies involve RNA isolation and further the expression analysis of the same using reverse transcription PCR (Polymerase Chain Reaction) and primers of different proteins expressed during cancer. This will also clearly demonstrate the effect of drug and changes made by it at the DNA level for different doses.

26. Toxicity Study • Herbo-mineral therapeutics are one of the most promising area of treating disease like cancer. The branch of Ayurveda which deals with medicinal properties of herbo- metallic and herbo- mineral preparations is known as RASASHASTRA and the drugs which are used in the branch known as RASA AUSHADHIS. They were more popular during the periods of the LORD BUDDHA due to faster relief, lesser and agreeable dose and mysterious efficacy as compare to only herbal drugs. So herbo-mineral drugs are considered more useful and effective as medicines for Cancer in many ways, such as prophylactic, palliative, curative and supportive. Rasayana therapy and its role in cancer management are being screened in almost all leading Ayurved research institutes. Polyherbal formulations based on Rasayana therapy described in CharakShamhita showed remarkable improvement in quality of life of various Cancer patients who were receiving chemotherapy, radiation and to overcome the side effects of chemotherapy and radiation such as hair loss, weight loss, stomatitis and zerostomia.

27. Toxicity Study • A dose response acute toxicity study with LAS01 showed that there was no mortality in mice which was observed for 72 hours. LAS01 for 90 days (Chronic Toxicity) did not show any concurrent on drug control in term of Haemoglobin percentage and TLC etc. based on the test parameters used. It appeared that the LAS01 did not significantly affect the Hepatic and Renal functions. It was observed that there are no symptoms of toxicity and it does not produce any degenerative changes of tissue damage to significant extent in the organ studied. • At the normal therapeutically equivalent dose and fivefold higher dose; none of the biochemical and GBP parameters showed any significant toxic changes. Hence the drug may be labeled as “Non Toxic”.

28. DNA Study • Expression analysis of several proteins expressed during CancerThe anti-cancer drug of LavanyaAyurveda Pvt. Ltd. LAS01 and LAS02 have been found effective through various experiments like MTT and Flowcytometery analysis, but the effect and change on the genetic and expression level has to be analyzed by studying the protein expressed by the genes related to cancer and modulation or mutation that bring these effects and changes can be analyzed so as to know the exact drug activity and pattern of action on various cancerous cells. The studies involve RNA isolation and further the expression analysis of the same using reverse transcription PCR (Polymerase Chain Reaction) and primers of different proteins expressed during cancer. The expected results would demonstrate the relation between the protein regulation and the disease progression. This will also clearly demonstrate the effect of drug and changes made by it at the DNA level for different doses.

29. Effect on CD4count and Viral Load • AIDS is the deadliest disease of the decade, for which unfortunately even after several advancement in biomedical sciences, no curative treatment is available as no drug is capable of separating and recovering the motif between retro-viral DNA of the host. A study has been undertaken to evaluate the role of our Anti-AIDS Herbo-Mineral Compound (LAA-1) for assessing antiviral activity includes CD3, CD4, CD8, CD4/ CD8 ratio and viral load parameters. The result of the study indicate that a significant rise in CD4 counts (p<0.05) and a significant decrease in viral load (p<0.05). The AyurvedicHerbo-mineral preparation LAA01 under test is not only a viridicidal (drug inhibiting the HIV replication) as is evident from decrease in HIV load, but the drug also enhanced immunity as is evident from increases in CD4 and CD8 counts and their respective percentage, which maintained the quality of life (QOL) of the patients.

30. Cancer Cell Line bank Lavanya is developing a Cancer Cell Line Bank to study • The Anti-cancerous activity of our Herbo- minerals used in various types of malignancies • To ensure its efficacy, • To ensure its safety • To ensure its non toxic property • Cell line study helps to ensure the efficacy of Lavanya’s treatment on other incurable diseases

31. Tissue Culture Lab • LavanyaAyurvedic Pvt. Ltd. has established a Tissue Culture Lab and two projects are submitted to state DST and Dept. of Biotechnology (GOI) for approval. • Production of Katuki project has already been prepared and in process of submission to Dept. of Medicinal Board for approval. • Musali project is in process and as per the protocol media has been prepared.

32. Our Major Achievement • It was found while treating thousands Cancer patients that metastasis of the disease even in 4th stage is completely checked up without any recurrence. • If the malignant mass or growth is not necrosed, it becomes dissolved and excreted out by various microcirculatory channels within 4 to 8 month (varying to severity and involvement of deeper Dhatu as bone) after taking LavanyaAyurvedic Therapy. • External malignant wounds are completely treatable by LavanyaAyurvedic Therapy. But in case of internal wounds which become necrosed need surgical removal, after that they could be healed by Ayurvedic Therapy even in 4th stage of disease. Radiation and Chemotherapy is not required at all.

33. Our Major Achievement • There are false believes about Bhasma prepared by heavy metals like Hg, As, Pb, Cu etc are very toxic to body. Lavanya has proven that if the Bhasma are prepared according to classical Ayurvedic texts and dose is proper administered. • While treating thousands patients with Ayurvedic rasa rasayana therapy there were no adverse effects of after effects were found in all patients and it was regular monitored clinically and hematologically (Liver & Renal function test). • AsthaSanskaritParad (Processed mercury) as described in Ayurvedic texts, most effective rejuvenator is first time prepared by only in Lavanya

34. Drug Standardization Drug Standardization on Modern Parameter, Acient parameter quality • The issue of Drug Standardization has ever been so important in respect of Drug Industry either on small level or on large scale industry. At LavanyaAyurveda, where a large number of Ayurvedic formulations of various herbal and herbo- mineral sources are prepared for in house day to day use for patients either attending the out patients’ department or admitted for their treatment at in patients level. The quality control of the formulations has a vital role. Normally the standards as laid down in Indian Pharmacopoeia (I.P.) and Ayurvedic Pharmacopoeia of India (A.P.I.) are taken into account for ascertaining the standard or herbal or herbo- mineral substances. Lavanya has always been engaged in formulating and preparing all of its formulations in strict compliance of classical methods, as well as IP and API standards, besides GMP and FDA rules and norms.

35. Drug Standardization • To comply with the standards or A.P.I. and I.P., the drug at first is graded for a better selection of raw material. Thereafter it is washed with running pure water to remove the dust and then dried well. There after it is grinded in Pharmacy department. Before it is passed on to the manufacturing dept., the raw drug is subjected for its physico- chemical analysis at Quality Control and Quality Assurance Division of LavanyaAyurveda which is a well equipped Laboratory. The drug confirming the standards of A.P.I. and I.P. are used in the manufacturing of various formulations either herbal or herbo- mineral.

36. Drug Standardization Different tests on the basis of Ayurvedic Pharmacopeia of India (API) are performed at Lavanya: (A) Descriptioni- Macroscopic ii- Microscopic(B) Identity, Purity and Strengthi- Foreign matter ii- Total Ash iii- Alcohol soluble extractive iv- Water soluble extractive v- Acid insoluble Ash vi- Loss on drying vii- pH(C) Assay/ TLC Constituents(D) Phytochemical analysis(E) Microbial limit test(F) Classical bhasma testing Rekhapurnatwa, Varitaratwa etc. are performed for metallic Bhasma.

37. Quality control Department • Quality Control (Q.C.) of a drug is as necessary for use in the treatment as a good food for proper physical and mental development. Lavanya keeps its eye on every point so that the raw drugs and finished products showed to be full of established quality parameters. For this purpose the source of procurement of raw material is very much important. Sorted only patients raw-material is procured which must be having sufficient chemical constituents as well as away from any contamination and decomposition. The stage between the raw drug and the final or finished product is method of preparation. Lavanya follows strict adherence to classical methods and no deviation at any level is permitted in preparing or purifying raw drugs. G.M.P. and F.D.A. rules help a lot in this direction which are invariably adopted and followed by the manufacturing section with the expert assistance of Drug Standardization and Quality control division.

38. Biosynthesis of Metal Based Plant Nanoparticles and its Anticancer Activity

39. INTRODUCTION • Nanotechnology research is interdisciplanary with physic, chemistry, biology, material science and medicine. • Plant are the rich source of various biocompounds in combination such as meniral, vitamin, alkylating agent, prophylineactic agent, protiens, fibres, flavanoids, phenolic compounds and so on. • Although an array physical and chemical method have been used to synthesized plant based silver nanoparticles.

40. OBJECTIVE • The objective of the present work is, formation of medicinal plants based Silver Nanoparticles and their Anticancer activity . Firstly we have prepared Nanoparticle of Curcuminlongawith the help ofsilver nitrate. • MATERIAL AND METHOD • The dried powdered of C. longa was used for extraction process. 10gm of dried powder was continuously extracted with ethanol. The extract was stored at -4c for further analysis. • SYNTHESIS OF SILVER NANOPARTICLES • A 10ml of Curcuminlongaextract was added into 90ml of aqueous solution of 1mM silver nitrate (AgNo3) for reduction of silver nitrate into Ag+ ion and keep at room temperature for upto 5 hours .After 20 minutes the colour of the solution changed into yellowish black indicating the formation of silver Nanoparticles .The bio reduced silver Nanoparticles solution was filtered and filtrate was measured using UV-Visble absorbance.

41. RESULT • UV-visible spectra analysis • The progress of the reaction between metal ions and extract were monitored by UV-Visible spectra of silver Nanoparticles in aqueous solution after diluting a small aliquot of 500µl of the sample with 1ml deionised water in different wavelengths i.e.,335,340,380, and 420nm and in different reaction times i.e.1,2,3,4 and 5 hours at 340nm. • The scanning graph obtained from UV-Visible spectrometry gives the range of particle at 340nm and its absorbance is 2.274 possibly the formation of curcuma silver Nanoparticles

42. Graphfrom UV-VIS Spectroscopy 320 340 360 380 414

43. CONCLUSION • Plant based Nanoparticles are new and exciting field of interdisciplinary Science, which make us to use plant in effective manner • FUTURE WORK • To check the anticancer activity of C. longaNanoparticles with the help of exploring the mechanism of its activity • Formation of other medicinal plant based Nanoparticles.

44. A novel drug delivery: Nanomedicine • Nanoparticles are promising tools for the advancement of drug delivery. However, the bio distribution of these nanoparticles is still imperfect due to the complex host's reactions to nano- and microsized materials and the difficulty in targeting specific organs in the body. At LavanyaAyurveda R & D center researches are going on to optimize and better understand the potential and limitations of nanoparticulate systems

45. Expertise in dosage forms Devlopstandred of dosage Ayuvedic Medicine, Herbs & not at develop. • At Lavanya Center different type of suitable dosage form are prepared, which are tested for its identity, purity and strength before human use. Arka, Capsule, Cream, Ghana Satwa, Lotion, Eye drop are frequently prepared

46. In Vitro Cancer Cell Line Study • Effect of LAS01 an AyurvedicHerbo-mineral compound on anti-proliferative activity and induction of Apoptosis in Human Breast Cancer MCF-7 Cell line. • For apoptotic induction study we used two apoptotic parameters F.actin ring positive cells and Vitronectin receptor positive cells using immune- cytochemistry. • Cells were cultured in standard situation using DMEM medium. • Cells were treated LAS01 along with a control group treated with DMSO. After 24 and 48 hours, of the cells were treated with F. actin and Vitronectin primary antibodies following incubation of secondary antibodies. • F. actin ring positive cells and Vitronectin receptor positive cells were observed in Lieca Microscope under florescent die using cell counter with cell morphometric analysis provided with Microscope.

47. In Vitro Cancer Cell Line Study • As study the compound shows induction of Apoptosis in carcinoma cell lines. • To find out better apoptotic result, experiments were repeated with higher dose 100, 200 and 300μg/ml of LAS01 in ethanol. • The result shows 9.26%, 13.31% and 17.68% in F. actin positive cells and 8.62%, 12.96 % and 16. 83 % in Vitronectin receptor positive cells higher as compared to control group. • All the doses did not show Necrosis in cells. • Growth of control group and Proliferation in treated cell were noted. It was found that in comparison to control group 48.6% less proliferation was there in LAS01 treated group at 300μg/ml dose MTT assay was also performed. The data suggest maximum inhibition about 48.6% in the treatment of 300 μg/ml as compared to control group. The treatment of 10, 20 and 50 μg/ml dose inhibit 16%, 21%, 33% proliferation respectively in control Group

48. Cell Cycle study • Our herbo- mineral drug LAS02 arrests the cancerous cells in cell cycle at G0/G1 phase, and prevented the entry of pre-cancerous stem cells from G0/G1 phase into G2, the subsequent proliferative stage and inhibits cancer cells from completing the cell cycle. In cell cycle analysis for COLO-205 and DU-145 cell lines with potent anti-cancerous compound LAS02 at doses of 300-600µg/ml in MTT assay showed inhibition of proliferation and decreased the viability of the cancers cells significantly. The percentage retention of the cells in cell cycle for COLO-205 at G0/ G1 phase was 73.07% at dose 300µg/ml of LAS02, as compared to 52.16% in the control after 24hrs. In cell cycle analysis for DU-145, treated with LAS02, cells that retained at G0/G1 phase were 79.28%, at a dose of 400µg/ml after 48hrs, as compared to control of 62.41%.The apoptosis observed, at 400µg/ml drug concentration was 43.51%. The result of study shows that LAS02 acts as a potent anti-cancerous compound by inhibiting proliferation as well as by inducing retention of cells in G0/G1 phase along with apoptosis significantly at in-vitro level. 

49. Publications 1. Toxicity of A Novel Herbo- mineral Preparation LAS01 On Human Cancer Cell Lines And Its Safety Profile In Humans and Animals Summary: In present study hematological and biochemical parameters found safe at different dose level in animal. In human volunteer at therapeutic dose level it is found safe at complete course of treatment. LAS01 is effective in human cancer cell line of cervical (Hela) and Breast Cancer Cell line (Adenocarcinoma MCF 07).

50. Publications 2. LAS01 Novel Herbo- mineral Ayurvedic Drug inhibits proliferation and induces the apoptosis in MCF-7 breast and Hela cervical cancer cell lines Summary: Results show inhibition and anti proliferative activity of compound in a dose dependent manner and maximum anti-proliferative activity about 77% in MCF-7 and 78% in Hela cells were observed in a dose of 500µg/ml. In case of anti apoptotic activity the results indicate maximum apoptotic inducing activity of 56.8% in 500 µg/ml dose in MCF-7 & 58.2 % in Hela VNR positive cells and 57.6 % in MCF-7 & 59.3 % in Hela F-actin cells respectively which was found to be significantly higher as compared to control group. The findings show a potent anti-proliferative and apoptosis inducing activity of compound LAS-01. The effects on cell proliferation exhibit a potent anti-proliferative activity of LAS01 and a comparable apoptotic inducing activity of various doses of LAS01 which suggests a strong in vitro anti-cancerous activity of this promising compound.