
==== Front
J Ayurveda Integr Med
J Ayurveda Integr Med
Journal of Ayurveda and Integrative Medicine
0975-9476
0976-2809
Elsevier

S0975-9476(24)00081-0
10.1016/j.jaim.2024.100966
100966
Review Article
Reverse pharmacology based clinical protocols for noninvasive integrative management of low grade cervical precancer lesions: Rationale and outcomes
Joshi J.V. a
Raut A.A. a
Paradkar P.H. prajakta.hsp@gmail.com
mrckhs@gmail.com
a⁎
Jagtap S.S. b
a Kasturba Health Society-Medical Research Centre, Vile Parle, Mumbai, India
b Ayurvidya Prasarak Mandal-Seth RV Ayurved Hospital, Sion, Mumbai, India
⁎ Corresponding author. Kasturba Health Society-Medical Research Centre, Vile Parle, Mumbai, 400056, India. prajakta.hsp@gmail.commrckhs@gmail.com
04 9 2024
Sep-Oct 2024
04 9 2024
15 5 10096630 6 2023
13 3 2024
5 5 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Prevention is the most efficient and cost-effective method to combat cervical cancer for which High Risk Human Papilloma Virus (HR-HPV) infection is identified as the major causative factor. HPV vaccination is for primary prevention whereas surgical ablation of precancer is for secondary prevention after HPV infection has occurred. Screening of women for early detection of Squamous Intraepithelial Lesions (SILs) with Papanicolou smear (Pap smear) is a desirable pre-requisite. Surgical ablation which invites invasive procedures is not accessible nor affordable to the larger section of the population. We propose here a non-invasive integrative management approach for the early phase of cervical pre-cancer.

In tune with the reverse pharmacology approach, ‘experience-exploration- experimentation’, we have conducted five clinical studies related to Turmeric extracts for chemo-preventive activity and non-surgical feasibility. We were able to achieve arrest or regression in Low-grade SILs in all 41 women participating in these studies.

The unique features of this integrative management approach were i) Avoidance of surgery-associated trauma, cost and complications ii) Standard of care for associated genital infections iii) Feasibility when surgery was not accessible iv) Scope for repeating the noninvasive treatment.
==== Body
pmc1 What is the purpose of this review article?

Cervical cancer, despite being preventable, is currently the second most common cancer in Indian women and is associated with a high morbidity and mortality [1]. The purpose of this article is to briefly present our research team's published data on the chemo-preventive potential of Curcuma longa (Turmeric) extracts at cervical pre-cancer stage. Chemoprevention is noninvasive and is always cost-effective as compared to the treatment of invasive cancer. It is also more humane because patients are spared radical surgery, or chemotherapy and radiotherapy. This clinical research conducted over two and a half decades is based on Reverse Pharmacology approach of ‘experience-exploration-experimentation’ [[2], [3], [4]]. The purpose is not to review the chemo-preventive action of individual components like curcumin or turmerone as there is extensive literature and several reviews available on these topics [[5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16]]. We wish to highlight and summarize here our experience of an integrative management approach in early cervical pre-cancer conditions. The integrative approach consists of pretreatment with antimicrobials for syndromic management of genital infections or Reproductive Tract Infections (RTIs) [17], followed by standardized turmeric extract for 10–12 weeks by oral route with the aim of achieving regression in Papanicolaou (Pap) smear abnormality.

The review is primarily based on published clinical data by the team on i) Healthy Volunteer safety study and organ function tests (N = 9) with Turmeric oil [18] ii) Demonstration of persistent Low - Grade Squamous Intraepithelial Lesions (LSIL) in Pap smears of women treated only with antimicrobial therapy (N = 15) [19] iii) Healthy volunteer study of oral standardized Super Critical Extract of Turmeric Oil (SCE-TO) for tolerability and organ function tests (N = 6) [20] iv) Clinical study on Integrative treatment with Antimicrobials (for associated genital infections) followed by SCE-TO for 12 weeks in women with a Pap smear Diagnosis of LSIL (N = 21) [20] v) A clinical post-treatment evaluation, including Pap smears, of the above study participants of SCE-TO treated women (N = 14) compared with control cases (N = 10), at 6–12 monthly intervals, up to 3 years [21] vi) Clinical study in a new group of women with LSIL with integrative treatment using Antimicrobials followed with a Holistic Turmeric extract (Curcumin + TO + Curcuma Polysaccharides) for 10 weeks (N = 20) [22]. With this integrative approach we observed success in all patients, either regression or arrest of the Pap smear abnormality.

Cervical cancer can usually be prevented if precancerous conditions are detected early and treated. Noninvasive or pharmacological methods of cervical cancer prevention have been tried earlier, however, these have been associated with insufficient efficacy or intolerable side effects [[5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16]]. We are not claiming a regression of high grade Squamous Intraepithelial Lesion (HSIL) which may also be possible, as reported in other studies, and that is a limitation of our studies. However, since LSIL is an early recognized precancerous condition [23], the results of these studies in terms of safety, side effects, ease of administration, benefit of treatment of associated infection and efficacy are encouraging.

2 Why is there a need for noninvasive chemoprevention of cervical cancer in the era of HPV vaccine?

Chronic inflammation with HR-HPV is causative for cervical cancer in more than 95% of cases. Currently very effective vaccines are available against the common carcinogenic HR-HPVs. However, HPV vaccines can prevent only up to 70–80% of cancers. Moreover, despite a recommendation by the Federation of Obstetric & Gynecological Societies of India (FOGSI) and Indian Association of Pediatricians (IAP), the vaccine coverage is <1 % in India. The public health vaccination program for girls may be initiated by the Government later in 2024, whilst, vaccine in open market is available but expensive for majority of women. Secondary prevention, i.e. after HPV infection has occurred, by screening with Pap smears/HR-HPV testing for detection and surgical management of precancerous conditions is also suboptimal [24]. Therefore, despite being almost 100% preventable, cervical cancer continues to ravage many Indian women. The recent global analysis of changes in cervical cancer trends showed decreased prevalence in many countries, however, in some countries including India, increased incidence was observed [1].

Secondary prevention is possible when low-grade, or high-grade precancerous conditions i.e. LSIL and HSIL, are detected during screening by Pap smears, colposcopies or Visual Inspection with Acetic Acid (VIA). The presence of HR-HPV test is rapidly becoming available in India but the costs are still prohibitive. In low-cost settings, surgical ablation treatment is advised based on HR-HPV or VIA positivity but ideally, treatment should be based on an additional Pap smear or a colposcopy [24]. The surgical treatment of SILs is very effective if carried out appropriately in short term studies. But long-term studies (>36 months) have shown a recurrence rate of 3–7% and some other complications [25]. Another consideration in a vast country like India is that the expertise for surgical ablation is not accessible or not affordable to many women.

Surgical ablations may unnecessarily create undesirable outcomes as described in Fig. 1.Fig. 1 Hurdles and undesirable outcomes of surgery for Squamous Intraepithelial Lesions. of Cervix observed in long term follow up.

Abbreviations: Govt = Government; Rx = Treatment; CS=Caesarian Section; FU=Follow up; HPV=Human Papilloma Virus *Unnecessary/Excessive – The surgical ablation may be unnecessary when it is based on ‘See & Treat’ approach when resources are poor, and is based only on VIA because VIA has a Low Specificity Rate.

Fig. 1

HPV test positivity indicates infection by the virus but does not show if cellular abnormality has developed. Hence if surgical ablation is done at the age of 25 years it may be unnecessary at that stage if ‘see and treat’ policy is based only on HR HPV positivity or only on VIA test. In these cases another test to confirm degree of abnormality is essential-what is known as ‘the triage of screening test’. Because the woman just has infection and may not have developed cancer for 1-2 decades.

Therefore, there is a need for a noninvasive method for management of SILs for secondary prevention of cervical cancer. Various agents have been studied in early clinical trials by other investigators for noninvasive management of cervical precancer such as Folate supplements, Zinc supplements, Green Tea extracts, Resveratrol, Curcumin, Retinoid analogues, and chemotherapeutic agents like Cisplatin, difluoromethylornithine (DFMO) etc. Most of these as mentioned earlier are partially effective, or have moderate to severe side effects leading to discontinuations, so that they cannot be used by relatively young and asymptomatic women [[5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16]]. Turmeric extracts were acceptable, except in few women who developed vaginal burning and pruritus which sometimes led to discontinuation, but were not a cause of worry both, to the participants or to the physicians. This can be a good option, especially for younger women who wish to preserve their child bearing function, to avoid complications of surgery, for women who don't have access to surgery. Moreover, organ function tests and routine clinical chemistry which were carried out before and after treatment did not show sub-clinical harm. Nevertheless, we suggest supervised integrated treatment.

3 What is reverse pharmacology?

Conventional classical pharmacology path for drug discovery and development begins from molecule to mice to man whereas in Reverse Pharmacology (RP) approach [3,4] it starts from man to mice to molecule. RP aims to bring forward the therapeutic wealth from traditional knowledge wisdom. RP is a discovery path for identifying novel drug action, which opens up the interface between-traditional healthcare knowledge-base, modern biomedical science-technologies and modern medical therapeutic approaches [2]. The RP approach includes accessing the experiential-hits, undertaking exploratory studies for identifying-leads and then conduct well organized experiments to recognize drug-candidates. The expected outcome of RP is to have safe, effective, economical and innovative pharmaco-therapeutics for better global health. This approach is spearheaded by KHS-MRC under the leadership of Dr Ashok Vaidya and is acknowledged globally. This approach has identified many leads for therapeutics in different fields-neurodegenerative diseases, arthritis & musculoskeletal disorders, diabetes mellitus & metabolic disorders, hepatitis & liver disorders, cancer & precancer conditions, cervicovaginal infections, and other diseases [2].

4 What are the concepts about inflammation and cancer in allopathy and in ayurveda?

The allopathic concepts on etiopathology of cancer have continuously evolved from the earlier Hippocratic postulation of imbalance of body humors (which is similar in some ways to the Ayurvedic imbalance of tridoshas) and the common former belief that ‘cancer is a painless causeless recurrent swelling’ which is incurable [26]. The scientific discoveries have later shown chromosomal abnormalities or mutations as the primary change with uncontrolled proliferation. Apart from acute chemical or radiation insults the role of chronic inflammation has recently been reemphasized again in mutagenesis with strong epidemiological and experimental data [27]. A recent analysis of preventable risk factors of cancers has confirmed the association of infections and chronic inflammation in carcinogenesis, specially, in cervical, oropharyngeal, gastric, colonic and other cancers [28]. The stronger causative association of viral infections like HPV, HSV, Ebstein-Barr Virus, HBV, and the role of immune suppression, is well known, but even chronic bacterial infection due to Helicobacter pylori is now shown to cause gastric cancer. Chronic inflammation with colonic anaerobic bacteria is a cofactor for colon cancer, whilst some species of Chlamydia trachomatis (CT) and Bacterial vaginitis (BV) are co-factors for cervical cancer [29].

Chronic and persistent infection with HR-HPV is a known cause of cervical cancer and HR-HPV test is positive in more than 95% of cervical cancers. That is the reason why HPV vaccine was developed and is recommended as the main policy for primary prevention of cervical cancer. HR-HPV infections can cause some other cancers also, and the vaccine is recommended in boys as well as girls. In our earlier studies we too had detected a statistically significant association of HPV and Bacterial Vaginitis cervico-vaginal infections with cervical neoplasia and cancer [30,31].

Ayurveda, a holistic healthcare system for disease management guides on identifying the causative factors, reversal of the etiopathogenesis, restoration of dosha homeostasis and optimization of the dhatus to restore the health. Giving nomenclature to a clinical condition has not been encouraged in Ayurveda [32]. However, obvious the clinical conditions which can be equated to cancer, especially solid tumors, in Ayurveda is Arbuda [[33], [34], [35], [36], [37]]. Arbuda are further classified based on the predominance of dosha (vataj, pittaja, kaphaj & tridoshaj), and primary involvement of a dhatu/tissues e.g. (mansajarbuda, medajarbuda, rakarbuda), adhishthan or localization e.g. (nasarbuda, galarbuda, karnarbuda, vartmarbuda, oshtharbuda, talvarbuda, sirarbuda) and stage of the disease e.g. (adhyarbuda-recurring, dwayarbuda-metastatic).

Some other clinical conditions which may be attributed to the neoplastic condition are asadhya-vrana/non-healing ulcer, asadhya-galaganda/thyroid tumor, asadhya gulma/abdominal tumors, asadhya kamala/hepatoma, asadhya pradar/refractory vaginal discharge-cervical cancer [38].

Ayurvedic concepts of Agni, Ama and Bala are most relevant to smoldering chronic inflammation. Agni a digestive-metabolic-bio-transformative capability when gets deranged, it produces Ama. Ama an undigested food substance when interacts with dosha-dhatu-mala and produces an auto-intoxic conglomerate. This auto-intoxicating substance disrupts the innate immunity (Bala) and facilitates pro-inflammatory environment in the biological system. Correcting the Agni, resolving the Ama and restoration of Bala is essential to overcome the chronic inflammation [39].

5 What was the non-invasive & integrative approach?

In earlier reported preventive trials for cervical cancer with chemoprevention (pharmacotherapy) or surgical ablation [[5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16],25], there was no mention of pretreatment with antimicrobials. Our previous studies on RTIs in women [30,31] and other studies had revealed a high prevalence of individual RTIs and multiple RTIs in young women, asymptomatic or with mild symptoms. A significant correlation was also reported with cervical precancer and cancer.

In view of the role of chronic inflammation in carcinogenesis, both in Allopathy and in Ayurveda as described above, we firmly believe that an antimicrobial treatment is necessary prior to the anticancer action of Turmeric. We know that Haridra itself is anti-inflammatory in many situations however evidence of its effect in specific RTIs or SILs is lacking. We therefore adopted the NACO guidelines [17] for syndromic treatment of RTIs, as the participants were asymptomatic or had minor symptoms in precancerous phase, and because specific diagnosis through multiple microbial cultures or other tests was not possible. We always discussed the role of RTIs in general health with the couple, and the chance of reinfections if both partners were not treated simultaneously, and insisted on abstinence or the use of condoms during RTI treatment for a total of 2 weeks. This was part of our participant consent form and pretrial counseling. Information to the couples that STDs are most often transmitted sexually, but non-sexual transmission through the use of common toilets, swimming pools, and intrauterine vertical transmission to the fetus is known, ensured co-operation from both partners for the syndromic treatment of associated genital infection even if they were asymptomatic or with mild symptoms like white discharge or burning in micturition. Occasionally surgical ablations may cause complications as given in Fig. 1, and the alternative of surgical ablation was included in the patient information sheet.

The noninvasiveness in this approach was avoidance of surgical ablation [25] whilst achieving regression of LSIL, which remained persistent even after discontinuation of treatment [21]. This approach also has the potential to repeat this integrated chemoprophylactic course if a recurrence of LSIL is observed in Pap smears during long term follow up. Both components, Antimicrobials and Turmeric extracts, are based on pharmaco-therapeutics. This approach is also accessible to a larger number of clinical facilities as training for surgery and surgical equipment are not essential. Thus, it can reach a wider population of women across the nation. Nevertheless, self-administration or unsupervised administration is not recommended.

The Integrative aspect was on including pretreatment with antimicrobials for possible associated genital infections so that the ‘inflammation’ was taken care of before starting the anticancer turmeric extracts. As per the National policy for management and control of RTIs we treated both partners, ie the couple, simultaneously so that reinfections were less likely, and as an added advantage, the complications of RTIs, which are a menace in themselves, were also likely to be prevented.

Based on literature review, our own experience of persistence of LSIL changes after antimicrobial treatment [19], we did not expect antimicrobial treatment alone to cause regression of SILs. In view of extensive literature on anticancer activity of turmeric extracts [5,8,[40], [41], [42], [43], [44], [45]] we decided to use Turmeric extracts for the purpose of achieving regression of LSIL.

6 What was the rationale for using oral turmeric extract and oil for this condition?

At our centre the team of trans-system experts had shown the anticancer activity of whole turmeric extract, curcumin and turmeric oil extract, and also isolated compounds like curcumin and turmerone, and in in-vitro and in-vivo experimental studies. There was a plethora of data from several authors on anticancer activity of turmeric extracts [[40], [41], [42], [43], [44], [45]] and individual components against various cancer cell lines and in in vivo models also. DNA protection was observed with turmeric extracts. An Ames’ test for mutagenicity was negative for curcumin as well as for turmeric oil. As most researchers from the rest of the world were concentrating on curcumin, Turmeric oil, a by-product during curcumin extraction, was usually being discarded or used for dermatological applications by some. Scientists from our centre studied the anticancer activity and safety of Turmeric oil (a mixture of turmerone, cineole, zigiberine etc.) in experimental and clinical studies.

At our centre exploratory clinical studies were undertaken on the use of oral turmeric oil (TO) capsules for reversal of Oral Submucous Fibrosis (OSMF), a precancerous condition for oral cancer [43,44]. The outcomes of these and verification of reduced DNA damage (micronuclei counts) after treatment encouraged us to explore the use of TO in another squamous epithelial cancer ie cervical cancer, which is a cause of high morbidity and mortality in women in India. Although OSMF is considered to be irreversible and progressive, the use of TO capsules for 3 months gave symptomatic relief and also showed objective improvement in mouth angle widening and reduction in buccal epithelial and peripheral lymphocyte micronuclei counts and in DNA damage. An organ function study in healthy human volunteers with supervised intake of the same formulation in a clinical pharmacology unit confirmed its clinical and biochemical safety [18].

Thereafter the first ever clinical trial on a turmeric oil extract in chemoprevention of cervical cancer in women with cervical SILs as detected by Papanicolaou smears and confirmed by colposcopy (to exclude high-grade lesions or cancer), was planned and undertaken subsequently [20].

Studies have confirmed a long precancerous phase between HPV infection and development of invasive cancer during cervical multistep carcinogenesis and this allows a window of opportunity to treat SILs during this phase for preventing invasive cancer [46].

Following the first clinical study on TO, there was further evidence of anticancer action of turmeric extracts, and turmerone [47] including experimental work in our centre [48,49]. We therefore continued to use the turmeric extract for integrative treatment but with the holistic extract instead of TO alone in the second clinical study [22].

7 What was the design of the first study of turmeric oil in LSIL?

Review of the literature at that time showed that curcumin was studied in precancerous conditions ie Squamous Cervical Intraepithelial Neoplasia with variable results [5,8,10]. We therefore planned a new study with TO standardized as shown below in Fig. 2.Fig. 2 Preclinical evaluation of SCE-TO Ref. [20](Supercritical extract of Turmeric oil).

Fig. 2

Briefly an experimental study was conducted in collaboration with the Advanced Centre for Treatment Research & Education in Cancer (ACTREC), Mumbai which showed that the extract was safe, and showed some preventive potential by experiments in nude mice treated with human xenograft cervical cancer implants and treatment with two doses of SCE-TO and compared with untreated controls. This was carried out pretrial (2008) but was published later (2020) due to an unrelated reason [50].

The earlier volunteer study [18] was with 0.2 ml TO capsules prepared by traditional fractionation method and contained about 35% of Tumerone. The new clinical study for LSIL regression was planned with a supercritical extract of Turmeric oil (SCE-TO), which yielded a higher concentration (>60%) of Turmerone, hence in addition to the essential preclinical testing a volunteer study was repeated in a hospital setting in healthy ambulatory adults with supervised intake of SCE-TO 0.2 ml capsules three times a day for 4 weeks. The formulation was well tolerated clinically with no adverse clinical or biochemical effects [20]. Subsequently, the clinical study on anticancer activity was undertaken in women undergoing routine Pap smear screening. The design of this study is depicted in Fig. 3.Fig. 3 Basic graphic design of the first clinical study in LSIL cases.

Abbreviations: SCE/TO= Supercritical extract of Turmeric Oil; Pap test = Papanicolaou test, wks = weeks; VIA=Visual Inspection with Acetic Acid; BD = Twice daily; LSIL = Low - Grade Squammous Intraepithelial Lesion; N/C=Nucleocytoplasmic ratio; SAE= Severe Adverse Effect.

Fig. 3

The protocol involved medical history, general physical and gynecological examination, Pap smear, VIA, colposcopy, and blood and urine tests to confirm that organ functions were within normal limits. This was followed by a single dose of antimicrobial kit (Secnidazole + Forcanazole + Azithromycin), as per National Guidelines for management of RTIs, given to both, the women and her spouse, who were also advised two weeks of abstinence (or use of condoms) to prevent reinfections. This was followed by SCE-TO capsules (0.2 ml) twice daily after breakfast and dinner for 12 weeks. The women came for scheduled clinic visits until the duration of treatment. Serum Interleukin-6 (IL-6) levels were measured in 14 cases before and after treatment and micrometry was performed on Pap smears for changes in Nucleocytoplasmic (N/C) ratio and other cytological abnormalities.

8 What was the outcome of the first study for cervical precancer?

Out of 1473 women screened for cervical cancer 88 women were detected to have LSIL in Pap smears. Out of these 21 women fulfilling enrolment criteria, gave informed consent for the study. In these women colposcopy and VIA were used to exclude high-grade lesions and invasive cancer. One enrolled case did not start treatment due to non-medical reasons. The antimicrobial treatment for genital inflammation was well tolerated by almost all couples. The SCE-TO treatment was also well tolerated systemically. However, one case discontinued due to local side effects of vaginal dryness, itching and burning within one week hence she was also excluded from analysis. Out of the remaining 19 cases another three had same local side effect, in tolerable limits, and they continued for four to twelve weeks. After 4–12 weeks of integrative management none of the 19 cases had progressed to higher grade or HSIL as observed by Pap smear and colposcopy; 16 cases showed regression to Atypia, Inflammation or normal Pap smear; Nucleocytoplasmic ratio decreased significantly in Pap smears after treatment, and serum IL-6 levels also declined significantly in these women [20]. After completion of treatment, women were advised a 6 monthly follow up with Pap smears for at least for 24 months.

We were able to follow up 14 participants up to 36 months after discontinuation of treatment. There was no recurrence of the LSIL. One woman, who had atypical cells reported in follow up, agreed for a second course of SCE-TO for three months again and had no side effects with complete regression to a negative Pap smear. A control group from the same period, ie women with LSIL who received only antimicrobial treatment, but did not participate with SCE-TO treatment, was also followed up (n = 10). Out of these, half cases had a Pap smear report of LSIL thus indicating the persistence of LSIL when SCO-TO treatment was not taken [21].

9 How was the second study different from the first one?

A subsequent publication showed that turmerone potentiated the anticancer activity of curcumin in vitro [47]. At our centre new experimental in-vitro and in-vivo studies documented various mechanisms of anticancer activity of different turmeric extracts and mixtures in HPV-negative and HPV-positive cervical cancer cell lines [48,49]. Moreover better anticancer effects were achieved with mixture of curcumin + turmerone + curcuma polysaccharides than with either curcumin or turmerone or polysaccharides alone, and anticancer mechanisms through antiproliferative, anti-inflammatory, immune modulation, anti-angiogenic and antimigration activities were elucidated. World literature also showed positive results of curcumin as a good adjuvant therapy to reduce side effects of radiotherapy and chemotherapy of invasive cervical cancer, and other cancers also.

Hence our second study was based on an oral formulation from a holistic turmeric extract. This was standardized to contain about 200 mgs of each component i.e., curcumin, turmerone and polysaccharides. The lipid soluble particles form of curcumin (LSPC) was used to improve bioavailability. This extract was also produced as per GMP and in a DST accredited laboratory with standard quality control. HPTLC standardization confirmed the contents initially, and after 3 years. The dosage schedule was similar to the previous study i.e., one capsule (600 mg) twice daily after breakfast and dinner for 10 weeks [22].

In order to reduce the duration of prophylactic treatment in relatively healthy younger women we also decreased the duration of this treatment to 10 weeks. Other items of the protocol like inclusion/exclusion criteria, confirmatory colposcopy prior to and during the trial, blood and urine tests, history, clinical examination, antimicrobial pretreatment to the couple, and frequency of follow up were similar to the previous study. Additionally, we tested bleeding time (BT) and clotting time (CT) before and after treatment [22]. In view of the discontinuation of one case in previous study for local vaginal side effects, we advised, as rescue medicine, milk or ghee as anupan as per Ayurvedic principles, only if women complained of side effects after starting the Holistic extract. We also included few women with well controlled Diabetes mellitus or hypertension if their condition was steady for previous 3 months and hence in combination with a shorter duration of treatment we could complete enrolment after screening 614 women.

10 What was the outcome of the second study?

Out of 614 women screened, 32 were detected with LSIL by Pap smears, and out of these 20 women agreed to participate in the study. Out of these 20, none progressed during the study period of 10 weeks after integrative treatment with antimicrobials and holistic turmeric extract ie 18/20 regressed, and 2 remained arrested. A six-month follow-up after discontinuation of treatment showed persistent beneficial effects in all who came for 6 monthly follow-ups, and there was no recurrence of LSIL. There were no adverse clinical or biochemical side effects [22]. Other investigators [10] had reported vaginal burning and dryness, or mild coagulopathy (without clinical bleeding) with 500 mg of vaginal curcumin whilst another earlier paper [8] of vaginal use of curcumin also reported local vaginal symptoms despite a combination with Aloe vera and Nimba. We did not observe significant changes in BT and CT or heavy menstrual bleeding. This may be because in the other studies, higher doses of curcumin were used and vaginal administration may have increased bioavailability due to the vascular by-pass effect. We have avoided local vaginal administration of turmeric extracts because of the poor acceptability of yellow discoloration of personal clothing due to vaginal discharge and the possibility of increased absorption due vaginal circulatory bypass effect. We also discussed with the vaginal route with contemporary Ayurvedic gynecologists and were informed that local application of haridra is not preferred in routine gynecological practice because it can cause aggravation of pitta in the form of vaginal irritation, burning and dryness, apart from the yellow color of discharge. Exceptions probably are mentioned when the arbuda is bleeding and in that case, locally extracts of haridra and mulak are described [35], whether as raktastambhak or for management of cancer is not clear from the texts.

One case discontinued after four weeks due to the same local side effects and another three had milder local complaints but continued the treatment. The addition of Anupan did not help materially in these cases. We believe that the use of lipid-soluble particle form of curcumin in the new formulation may have increased the bioavailability of curcumin to an extent that side effects (Pittaprakopa) were manifested. We have discussed this in the published report by postulating that any doshaprakopa manifests earlier in the dushit or pathologically affected part and since in these cases there is cervico-vaginal infection, the pitta prakopa symptoms appeared in vagina even though the administration was oral. Nevertheless, it was satisfying to observe by Pap smears and by colposcopy that 18/20 cases responded to oral treatment with regression.

11 What was the third aborted study?

Given the encouraging findings in the third year of the second study, we also planned a feasibility study to see if we could have the same protocol but with lesser clinical visits and fewer research inputs. However, we could enroll only three cases due to the onset of the Covid-19 Pandemic, and the single case who could come for follow-up had Pap smear regression and did not have any side effects. This study was discontinued and Ethics Committee was informed.

12 What is the need for further study and how will it differ from previous studies?

The previous two studies in Low-Grade SIL cases, treated with antimicrobials (single dose) followed by oral turmeric extract for 10–12 weeks indicate that there was regression of LSIL in 36/41, and arrest in 5/41 cases and that noninvasive integrative method of chemoprophylaxis of cervical cancer is a potential alternative to conventional surgical ablative surgery in LSIL. The estimated number of women with LSIL, a precancerous condition, is always a hundred times more than invasive cancer cases in the population. In view of the paucity of trained gynecologists/paramedical staff for ablative procedures all over India, we hope that this method has a future in the management of these conditions though further evaluation is desirable. This method also has the advantage that it has the potential to be safely repeated if there is the recurrence of the SIL. Since surgery is avoided, there is no interference in follow-ups with Pap tests or VIA because there is no issue of post-surgical cicatrization.

The recent mega-data from global surveys [1] clearly shows the limited availability of HPV vaccine, inadequate screening and deficient management of HPV lesions in many countries. Consequently cervical cancer incidence has increased in countries with limited resources, including India. It may be a decade before all eligible girls/women are vaccinated in India. Until then we need to use all available options for disease prevention.

We would therefore like to pursue this method in a larger number of cases, and with the following improvisation: in 5/41 women, LSIL was arrested but did not disappear, though the inflammation and number of abnormal cells in PAP smears had reduced. Moreover, although there was a regression, few cases had local side effects. We believe this result can be improved by adding to the Integrative approach a local application of Ayurvedic formulation, which has anticancer activity and which may at the same time counteract the aggravation of Pitta dosha as per Ayurvedic principles. The local application can be reduced to a limited number of postmenstrual days and need not continue daily throughout the 10 weeks so that the acceptability of the prophylactic chemotherapy is not compromised.

13 Challenges for the future study

Earlier studies were conducted in small budget primarily because the Papanicolaou screening and colposcopy were conducted free of cost by one of the investigators. The formulations were standardized and provided for research free of cost. The clinic assistant also worked on a voluntary basis once a week with minimal compensation. No volunteer travel compensation was available to the participants in the small budget and we depended on pretreatment counseling for all follow ups. For the future studies we would like to make provision for all these shortcomings to improve the success rate and a near 100% long-term post-treatment follow-up up till at least 2–3 years. The cost of standardization and production of formulation for local treatment will also be an added cost even if the oral formulation is provided free of cost. An exploratory study can be undertaken but a controlled study is desirable. Since randomization and blinding is not possible, the control group can be obtained by parallel enrollment in 2 different hospitals with identical protocols except for the treatment regime. The number of cases and budget will increase proportionately if a controlled study is approved. HR-HPV tests are expensive in India and are not included in the algorithm in National Guidelines even now because of the non-availability and the cost especially in rural areas [24] but the test has been recommended by the ICMR Expert group. This test should be included at least once before starting treatment.

International guidelines for follow-up of LSIL vary from country to country being appropriate for well-developed countries where resources are adequate but may be compromised in countries with restricted resources [24,51].

The unique feature of this approach is that all guidelines suggest a conservative follow-up of LSIL with a Pap smear after 6 months and since there is no active treatment in the intervening period the integrative, non-invasive approach proposed in this review is worthwhile to achieve regression in precancer without compromising any guidelines.

HPV vaccination is available in cities in the private sector and is being currently incorporated in the National program in India. It is known that all LSIL cases may not be HR-HPV positive, as the current tests detect only 2 to 9 HRHPV types, and hence the available protective vaccine can be taken by women up to 26 years of age. We would like to include information in the Informed Consent Document about the vaccines and their limitations and availability to adult women participants. This would ensure added cervical cancer protection for those can afford and who agree to the vaccination. This may be advised after the completion of 3 months of active treatment period so that the study results are not vitiated.

Besides being cost-effective, prevention of cancer is more humane for the patients as there is less pain, less suffering, and greater accessibility compared to the treatment of invasive cancer, and is associated with a much better quality of life than cancer survivors.

Looking at the success of this integrative approach involving counseling of the patient for prevention of cancer we believe that this is a feasible strategy, at all women's healthcare facilities.

Funding sources

None

Declaration of generative AI in scientific writing

None used.

Author contributions

Joshi JV: Conceptualization, Formal analysis, Writing – originaldraft, Writing – review and editing, SupervisionRaut AA: Writing – original draft, Writing – review and editing, SupervisionParadkar PH: Data curation, Writing – review and editingJagtap SS: Data curation, Writing – review and editing

Conflict of Interest

None

Acknowledgements

We are grateful to Prof Ashok DB Vaidya, for his mentorship. We also appreciate the institutional support from Ayurvidya Prasarak Mondal, Sion, Mumbai and Kasturba Health Society, Sevagram, Wardha.

Peer review under responsibility of Transdisciplinary University, Bangalore.
==== Refs
References

1 Global Burden of Disease Cancer incidence, mortality, years of life lost, years lived with disability, and disability-adjusted life years for 29 cancer groups from 2010 to 2019. A systematic analysis for the global burden of disease study 2019 JAMA Oncol 8 2022 420 444 34967848
2 Vaidya A.D.B. Integrative vision in cancer research, prevention and therapy J Ayurveda Integr Med 15 1 2024 100856 10.1016/j.jaim.2023.100856
3 Vaidya A.D.B. Reverse pharmacological correlates of ayurvedic drug actions Indian J Pharmacol 38 2006 311 315
4 Raut A.A. Chorghade M. Vaidya A.D.B. Patwardhan B. Chaguturu R. Reverse pharmacology, innovative approaches in drug discovery: ethnopharmacology, systems biology and holistic targeting 2017 Academic Press 89 126
5 Cheng A.L. Hsu C.H. Lin J.K. Hsu M.M. Ho Y.F. Shen T.S. Phase I clinical trial of curcumin, a chemopreventive agent, in patients with high-risk or pre-malignant lesions Anticancer Res 21 2001 2895 2900 11712783
6 Abu J. Batuwangala M. Herbert K. Symonds P. Retinoic acid and retinoid receptors: potential chemopreventive and therapeutic role in cervical cancer Lancet Oncol 6 2005 712 720 16129372
7 Sasieni P. (Review) Chemoprevention of cervical cancer Best Pract Res Clin Obstet Gynaecol 20 2006 295 305 16386963
8 Basu P. Dutta S. Begum R. Mittal S. Dutta P.D. Bharti A.C. Panda C.K. Biswas J. Dey B. Talwar G.P. Das B.C. Clearance of cervical human papillomavirus infection by topical application of curcumin and curcumin containing polyherbal cream: a phase II randomized controlled study Asian Pac J Cancer Prev APJCP 14 2013 5753 5759 24289574
9 Garcia F.A. Cornelison T. Nuño T. Greenspan D.L. Byron J.W. Hsu C.H. Alberts D.S. Chow H.H. Results of a phase II randomized, double-blind, placebo-controlled trial of Polyphenon E in women with persistent high-risk HPV infection and low-grade cervical intraepithelial neoplasia Gynecol Oncol 132 2014 377 382 24388920
10 Gattoc L. Frew P.M. Thomas S.N. Easley K.A. Ward L. Chow H.S. Phase I dose-escalation trial of intravaginal curcumin in women for cervical dysplasia J Clin Trials 9 2017 1 10
11 Ren B. Kwah M.X. Liua C. Zhaowu Shanmugam MK. Ding Lingwen Xiang Xiaoqiang Paul Chi-Lui Ho b Lingzhi Wang c f Pei Shi Ong b Goh Boon Cher Resveratrol for cancer therapy: challenges and future perspectives Phytomedicine 55 2019 269 281 30668439
12 Paulraj F. Abas F. Lajis N. Othman I. Naidu R. Molecular pathways modulated by curcumin analogue Diarylpentanoids Cancer. Biomol 9 2019 270 10.3390/biom9070270
13 Hassanzadeh K. Buccarello L. Dragotto J. Mohammadi A. Corbo M. Feligioni M. Obstacles against the marketing of curcumin as a drug Int J Mol Sci 21 18 2020 6619 10.3390/ijms21186619 32927725
14 Moballegh N.M. Abadi B. Poormoghadam D. Zarrabi A. Keyhanvar P. Khanbabaei H. Curcumin delivery mediated by bio-based nanoparticles: a review Molecules 25 3 2020 689 10.3390/molecules25030689 32041140
15 Zuo H.L. Huang H.Y. Lin Y.C. Cai X.X. Kong X.J. Luo D.L. Enzyme activity of natural products on cytochrome P450 Molecules 27 2 2022 515 10.3390/molecules27020515 35056827
16 Rodríguez C.P. Parween S. Pandey A.V. Bioactivity of curcumin on the cytochrome P450 enzymes of the steroidogenic pathway Int J Mol Sci 20 18 2019 4606 10.3390/ijms20184606 31533365
17 National Guidelines on Prevention Management and control of reproductive Tract infections and sexually transmitted infections 2014 Department of AIDS Control, Ministry of Health and Family Welfare Government of India
18 Joshi J.V. Ghaisas S.D. Vaidya R.A. Kamat D.V. Bhagwat A.N. Early clinical safety study with turmeric oil (Curcuma longa oil) in human volunteers J Assoc Phys India 51 2003 1055 1060
19 Joshi J.V. Affandi M.Z. Amin P. Vaidya R.A. Shah R.H. Persistence of cytologic abnormality after treatment of bacterial, parasitic and fungal infections in older women with low grade squamous intraepithelial lesion (letter) Acta Cytol 54 2010 242 243 20391993
20 Joshi J.V. Paradkar P.H. Jagtap S.S. Agashe S.V. Soman G. Vaidya A.B. Chemopreventive potential & safety profile of NBFR-03 (supercritical Curcuma longa extract) in women with cervical low- grade squammous intraepithelial neoplasia in Papanicolaou smears Asian Pac J Cancer Prev APJCP 12 2011 3305 3311 22471471
21 Joshi J.V. Paradkar P.H. Jagtap S. Paradkar H. Walwatkar P. Soman G. Persistent chemopreventive action of integrative treatment of low-grade cervical intraepithelial neoplasia- case series J Ayurveda Integr Med 7 2016 109 112 27475746
22 Joshi J.V. Jagtap S.S. Rastogi N. Walwatkar P. Nabar N.S. Hingorani L. Integrated non-invasive management of cervical low-grade squamous intraepithelial lesions observed in Papanicolaou smears with antimicrobials followed by oral Curcuma longa extract Asian Pacific J Cancer Biol 5 2020 89 97
23 Nayar R. Wilbur D.C. The Pap test and bethesda 2014 Acta Cytol 123 2015 271 281
24 FOGSI GCPR Screening & Management of Preinvasive Lesions of CervixVaccination H.P.V. Good clinical practice recommendations by federation of obstetrics & gynecology of India 2018
25 Martin-Hirsch P.P. Paraskevaidis E. Bryant A. Dickinson H.O. Surgery for cervical intraepithelial neoplasia Cochrane Database Syst Rev 2013 2013 CD001318 24302546
26 Di Lonardo A. Nasi S. Pulciani S. Cancer: we should not forget the past J Cancer 6 1 2015 29 39 10.7150/jca.10336 25553086
27 Balkwill F. Mantovani A. Inflammation and cancer: back to Virchow? Lancet 357 9255 2001 539 545 11229684
28 GBD 2019 Cancer Risk Factors Collaborators The global burden of cancer attributable to risk factors, 2010–2019: a systematic analysis for the Global Burden of Disease Study 2019 Lancet 400 2022 563 591 35988567
29 D'Antonio D.L. Marchetti S. Pignatelli P. Piattelli A. Curia M.C. The oncobiome in gastroenteric and genitourinary cancers Int J Mol Sci 23 17 2022 Aug 26 9664 10.3390/ijms23179664 36077063
30 Joshi J.V. Mali B.N. Hazari K. Shah R.S. Chitlange S.C. The association of cervical intraepithelial neoplasias and sexually transmitted diseases in Papanicolaou smears (correlation with cervical biopsy in a subset) J Vivek Inst of Med Sciences 17 1994 9 13
31 Joshi J.V. Mali B.N. Bhave G. Wagle U. Cervical neoplasia and cytological manifestations of sexually transmitted infections in HIV seropositive prostitutes. (Letter) Diagn Cytopathol 4 1993 63 64
32 Acharya Y.T. Charaka Samhita of Agnivesha Sootra sthana; Trishothiya: chapter 18, verse 44-46 vol. 108 2005 Chaukhamba Surbharati Prakashan Varanasi
33 Samhita Sushruta Ayurveda tatva sandeepika ed ambika datta shastri 2021 Chaukhambha Sanskrit Sansthan Publications Varanasi 350 420 [Chapter 11]
34 Ashtang-Hriday VagBhatt GK Garde Chaukhambha Sur Bharati Prakashan, chapter 30 2019
35 Tewari P. Chapter 7. Arbuda or tumours and cysts of the reproductive system Ayurvedeeya Prasuti-Tantra evam stri-roga second ed. 2000 Chaukhambha Orientalia Varanasi 385 395 Reprinted 2018
36 Ram Manohar P. Descriptions and classification of cancer in the classical ayurvedic texts IJHS 50 2015 187 195 10.16943/ijhs/2015/v50i2/48234
37 Rawat N. Roushan R. Management of arbuda (tumor) through multiple Ayurveda treatment modalities IJBAR 8 2018 639 644
38 Prasad G.C. Sahu M. Deshpande P.J. Concept of cancer in Ayurveda Ancient Sci Life 1982 172 176
39 Sumantran V.N. Tillu G. Cancer, inflammation, and insights from Ayurveda eCAM 2012 11 10.1155/2012/306346 306346
40 Nagbhushan Bhide S.V. Antimutagenicity and anticarcinogenicity of turmeric (Curcuma longa Linn) J Nutr Growth Cancer Inst 73 1987 737 741
41 Azuine M.A. Bhide S.V. Chemopreventive effect of turmeric against stomach and skin cancers induced by chemical carcinogens in Swiss mice Nutr Cancer 17 1992 77 83 1574446
42 Mehta K. Panayotis P. McQueen Aggarwal BB. Antiproliferative effect of curcumin (diferuloylmethane) against human breast tumor cell lines Anti Cancer Drugs 8 1997 470 481 9215611
43 Hastak K.A. Lubri N. Jakhi S.D. Effect of turmeric oil and turmeric oleoresin on cytogenetic damage in patients suffering from oral submucous fibrosis Cancer Lett 116 1997 265 269 9215873
44 Hastak K. Jakhi S.D. More C. John A. Ghaisas S.D. Bhide S.V. Therapeutic response to turmeric oil and turmeric oleoresin in oral submucous fibrosis patients Amala Res Bull 18 1998 23 28
45 Aggarwal B.B. Yuan W. Li S. Gupta S.C. Curcumin-free turmeric exhibits anti-inflammatory and anticancer activities: identification of novel components of turmeric Mol Nutr Food Res 57 2013 1529 1542 23847105
46 Weinberg R.A. Multistep tumorigenesis. Chapter 11 The biology of cancer 2007 Garland Science 399 462
47 Yue G.G. Chan B.C. Hon P.M. Lee M.Y. Fung K.P. Leung P.C. Evaluation of in vitro anti-proliferative and immunomodulatory activities of compounds isolated from Curcuma longa Food Chem Toxicol 48 2010 2011 2020 10.1016/j.fct.2010.04.039 20438793
48 Paradkar P.H. Dandekar S.P. Joshi J.V. Amonkar A.J. Vaidya A.B. Assessment of in vitro - in vivo antimigratory and anti-angiogenic activity of Curcuma longa Linn. and Tinospora cordifoliaWilld. Extracts in cervical cancer J Pharm Sci Rev Res 42 2017 87 93
49 Paradkar P. Dandekar S. Joshi J.V. Amonkar A.J. Vaidya A.B. Synergistic anticancer activity of the medicinal plant bioactives: curcuma longa linn. And tinospora cordifoliaWilld Cervical Cancer Int J Pharm Sci Rev Res 42 2017 151 160
50 Paradkar P.H. Juvekar A. Barkume M. Amonkar A.J. Joshi J.V. Soman G. Vaidya A.B. In vitro and in vivo evaluation of a standardized haridra (Curcuma longa Linn) formulation in cervical cancer J Ayurveda Integr Med 12 2021 616 622 10.1016/j.jaim.2021.06.002Linn 34531090
51 WHO guideline for screening and treatment of cervical pre-cancer lesions for cervical cancer prevention second ed. 2021 World Health Organization Geneva
