Elderberry extract and its use in the prevention or in the treatment of tumours

Abstract

The present invention relates to a total dry extract of elderberry (Sambucus nigra) and to its use as an anti-cancer drug both preventive and for treatment. In particular, the invention relates to an extract comprising active ingredients capable of effectively and synergistically combatting the growth of cancer cells totally regressing, in particular solid cancers.

Description

“ELDERBERRY EXTRACT AND ITS USE IN THE PREVENTION OR IN THE TREATMENT OF TUMOURS”DESCRIPTIONTECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates to a total dry extract of elderberry and its use as an anti-cancer drug both preventive and for treatment of tumours. In particular, the invention relates to an extract comprising active ingredients capable of effectively and synergistically combatting the growth of cancer cells by totally regressing solid cancers. STATE OF THE PRIOR ART

[0002] Cancer is considered the second leading cause of death in the world. Although there is a strong focus in its management, the scenario seems to take longer for both possible therapies and the control of its complications. To date, surgery and chemotherapy are the leading strategy as pertains to its control. However, patients diagnosed with cancer and receiving chemotherapy experience numerous side effects. Therefore, many people rely on complementary and alternative medicine.

[0003] Cancer therapies are associated with various challenges, including the onset of multi-resistant cancers, toxicological problems, severe side effects and financial burdens. To counter these effects, natural substitute products and adjuvant therapies are increasingly available which have received considerable attention for their safety, efficacy and economic aspects. Several pre-clinical and clinical studies have revealed that natural products and their combination with chemotherapeutics mediate their antitumor effects by modulating various factors, that is signalling pathways involved in promoting apoptosis, inhibiting excessive cell proliferation and mobilising the immune system.

[0004] Many bioactive substances present in phytocompounds have been identified as potentially active, although there are still few studies on the subject and need further study. For example, concomitant use of phytotherapics and chemotherapeutics has been shown to be useful in blood cancers but there are no scientific publications showing their usefulness on solid cancers.

[0005] In particular, elderberry with its twenty species (Sambucus Racemosa, Sambucus Canadensis, Sambucus Mexicana, Sambucus javanica), especially the European botanical species called Sambucus nigra L., is a plant known to have various therapeutic properties generally linked to anti-inflammatory and antioxidant activities. The parts of theplant that are considered relevant for this purpose are berry and flower extracts. As a matter of fact, these extracts are rich in sugars, particularly glucose and fructose, fibres including pectin, pectic acid, protopectin and cellulose. Both fruits and flowers also contain proteins which include sixteen amino acids, nine essential acids found in larger amounts in the leaves. The main amino acids are glutamic acid, aspartic acid and alanine. Fatty acids, on the other hand, are predominant in seeds and are basically polyunsaturated, including a-linolenic, linoleic and oleic acid. A high concentration of citric acid was also found, especially in fruits. In addition, essential oils have been identified in both berries and flowers, while both a-tocopherol and y-tocopherol have been found in seed flour. However, the most therapeutic substances are polyphenols, including flavonoids known for their antioxidant activity, such as for example chlorogenic acid, quercetin, rutin, isoquercetin, astragalin. Among polyphenols, there are high concentrations of anthocyanins in berries used in the food industry as colorants.

[0006] In addition, elderberry also contains some potentially harmful substances especially in the bark, leaves and petioles, for example cyanogenic glycosides such as sambunigrin, prunasin, zierin and olocalin. Other toxic compounds are lectins, some of which have N- glycoside activity.

[0007] Generally, berry or dried flowers extracts are mostly used as remedies for antiinflammatory, antipyretic, antibacterial, laxative, diuretic activities, especially in herbal preparations. There has been shown a certain anti-inflammatory, antiviral therapeutic activity, as well as improvement in the metabolism in some subjects (Bioactive properties of Sambucus nigra L. As a functional ingredient for food and pharmaceutical industry, Karolina Mlynarczyka et al., Journal of Functional Foods 40 (2018) 377-390). Treatments include the administration of an amount of dry extract from berries and flowers of 1 mg, or extract at the concentration of 252 to 3600 pg / ml, or even aqueous leaf extracts or flower extracts in capsules.

[0008] Among the data available in the literature, there is no evidence of the efficacy of elderberry extracts for the treatment of cancers. Although their potential antioxidant activity suggests their use as anticancer agents, there are no in vivo data to demonstrate their efficacy, particularly on solid cancers. The lack of experimental data on humans or animals, as well as side effects such as nausea, vomiting and diarrhoea due to overdose of berries, to date has not led to any datum on the effectiveness of elderberry in the treatment or prevention of cancer diseases.SUMMARY OF THE INVENTION

[0009] Therefore, the technical problem underlying the present invention lies in providing total elderberry extracts, preferably of the European Sambucus nigra L. species, for the treatment or prevention of solid cancers.

[0010] This problem is solved by a total dry extract specifically from elderberry petioles and bark that can effectively treat patients with solid cancer diseases. In particular, effective treatment of different solid cancers in different organs with complete remission years later, that is full recovery, has been observed.

[0011] Therefore, a first object of the present invention is a total dry extract of elderberry for use in the treatment or prevention of solid cancers.

[0012] A second object is a pharmaceutical composition comprising an anti-tumour combined with the total dry extract of elderberry.

[0013] A yet further object is a pharmaceutical composition comprising total dry extract of elderberry combined with a total dry extract of pomegranate.

[0014] Further characteristics and advantages of the total dry extract of elderberry of the invention will become more apparent from the following description of some embodiments provided purely by way of non-limiting example.DETAILED DESCRIPTION OF THE INVENTION

[0015] As explained in the introductory part of the present description, elderberry extract and in particular the extract from the European Sambucus nigra L. species contains a variety of substances among which carbohydrates, proteins, fats, fatty acids, organic acids, minerals, vitamins, essential oils and in particular polyphenols, which are known for their anti-radical (antioxidant) activity, are found in higher quantities.

[0016] According to the present invention, it has been found that different parts of the plant contain such substances in different concentrations but also with different compositions. In particular, it has been found that leaf petioles and bark are parts of the plant in which the phenolic active ingredients are most present in a specific combination of polyphenols, anthocyanins, glycoside phenols and lectins, the latter two compounds being potentially toxic. This combination would be responsible for a surprisingly synergistic effect such to enhance the therapeutic activity of the active ingredients known for in vitro antiinflammatory, antipyretic, antibacterial, laxative, diuretic activities. As a matter of fact, without being bound to any theory, the sap that reaches the bark and the petioles probably has a different composition compared to that of flowers and fruits or it is subjected to different processes.

[0017] Therefore, the pharmaceutical composition of the invention comprises a total dry extract of elderberry petioles and bark. In particular, the extract comprises a mixture ofpolyphenols, glycoside phenols, anthocyanins and [3-carotene. Among polyphenols, the most common are 4-caffeoylquinic acid, 3-caffeoylquinic acid, caffeic acid, catechin, epicatechin, rutin, 3-coumaroylquinic acid, isorhamnetin-3-rutinoside, ferulic acid, kaempferol-3 rutinoside, 1 ,5-dicaffeoylquinic acid. The most common glycoside phenols are sambunigrin, prunasin, zierin and olocalin. Anthocyanins are in particular cyanidin-3- sambubioside, cyanidin-3-glucoside, cyanidin-3-monoglycoside and cyanidin-3,5- diglycoside.

[0018] The polyphenol mixture is present in amounts preferably comprised between 4,800 ppm and 10,900 ppm on the dry weight of the total extract of petioles and bark. More preferably, the amount of polyphenols is comprised between 6,500 ppm and 8,500 ppm.

[0019] Glycoside phenols are preferably present in amounts of 5,000 to 20,000 ppm on the total dry weight of the extract.

[0020] Anthocyanins are preferably present in amounts of 300,000 ppm on the total dry weight of the extract.

[0021] [3-carotene is present in amounts ranging from 5 ppm to 12 ppm on the total dry weight of the extract.

[0022] The total dry extract of the petioles and bark is obtained according to the following protocol.

[0023] After collecting bark pieces from the trunk and branches of the plant and separating the petioles from the leaves, the harvest is washed under running water. Subsequently, both the pieces of bark and those of petioles are crushed up to obtaining parts measuring between 2 and 3 cm in size. These crushed parts are subsequently heated to 60 °C in a hydroalcoholic solution as solvent for 45 minutes. At the end of this treatment, there is obtained a liquid with a high water content in which there are dissolved the above- mentioned active ingredients contained in the bark and in the elderberry bark and petioles.

[0024] In a conventional low-temperature vacuum oven, the aqueous extract is heated to evaporate the water so as to obtain a concentrated soft extract, that is a product of paste consistency, similar to honey, which may range from 20 to 70 °Bx.

[0025] In order to reduce the bacterial load, yeasts, moulds and any other microbial agents present in the plant, the soft extract is subjected to a pasteurisation cycle at 80 °C for 4 hours. At this point, the extract is added with a pharmaceutically acceptable excipient and the whole is mixed and sprayed into a dryer where it undergoes dehydration and reduction into particles measuring from 200 to 500 microns in size.

[0026] The dry extract thus obtained is sieved to remove impurities and to obtain a homogeneous powder, before being encapsulated and packaged in blisters.

[0027] For the purpose of verifying the content of active substances, the elderberry petioles and bark, for example of the Sambucus nigra L. species, were characterised for the polyphenol content using the Orbitrap qExactive mass spectrometer and for the [3- carotene content by means of HPLC-UV.

[0028] The samples analysed were collected in April 2022. After collection, the petioles were separated manually from the leaves and the bark from the branch. An amount of petioles and bark was dried at 40°C. The main phenolic compounds present in the Sambucus nigra L. bark and petioles are listed in Table 1 and Table 2, respectively, and they all refer to dry weight. The composition of phenolic acids varies greatly depending on whether they are bark or petioles. Extraction protocols are for example those described in “Bioactive Compounds from Elderberry: Extraction, Health Benefits, and Food Applications” (Oana-Elena Pascariu and Fiorentina Israel-Roming, Processes 2022, 10, 2288, https: / / doi.Org / 10.3390 / pr101 12288).TABLE 1TABLE 2

[0029] In petioles, the most abundant molecules include 4-caffeoylquinic acid, 3- caffeoylquinic acid and kaempferol-3 rutinoside. Elderberry bark is rich in kaempferol-3 rutinoside and ferulic acid.

[0030] Furthermore, the total dry extract of Sambucus nigra L. petioles and bark may also comprise a part of the total dry extract of Sambucus nigra L. berries and / or flowers. This extract is obtained with a process identical to the one defined above for the dry extract of bark and petioles.

[0031] According to a preferred embodiment of the invention, the pharmaceutical composition comprises the total dry extract of elderberry combined with the total dry extract of pomegranate. This extract comprises anthocyanins, glucose, ascorbic acid, ellagic acid, gallic acid, caffeic acid, catechin, EGCG (epigallocatechin gallate), quercetin, rutin, minerals including iron, amino acids.

[0032] The total dry extract of pomegranate is obtained from various parts of the plant preferably it is obtained from the whole fruit alone using the same process as elder berries.

[0033] An example of substances that can be found in pomegranate extract are reported in table 3 below.TABLE 3

[0034] According to a further embodiment, the pharmaceutical composition comprises a total dry extract of Sambucus nigra L. combined with or separately with a synthetic anticancer substance for the treatment of solid cancers. Preferably the anti-cancer substance is enanthone (leuprorelin acetate), but other anticancer agents may also be administered for example including Docetaxel, Folfiri (Leucovorin, fluorouracil, irinotecan), Oxaliplatin, Capecitabine, Gemcitabine, monoclonal antibodies (Bevacizumab, Cetuxima, Trastuzumab, Panitumumab).

[0035] The pharmaceutical composition of the invention was successfully tested in cases of solid cancer in which the treatment provided for the administration of the total dry extract of Sambucus nigra L. petioles and bark at a dosage comprised between 300mg and 1500mg once or twice a day for 1 -10 months. Preferably the dosage ranges from 500 mg to 1000 mg once or twice per day for 3-6 months.

[0036] Preferably, the composition also comprises the berry extract administered in an amount comprised between 100 mg to 600 mg once or twice per day for 1 -10 months; more preferably between 150 mg and 300 mg once or twice per day for 3-6 months.

[0037] Should the composition also comprise the total dry extract of pomegranate, it is administered in amounts comprised between 100 mg and 600 mg (including ranging between 10 mg and 100 mg ellagic acid) once or twice per day for 1 to 10 months. Preferably the dosage ranges from 150 mg to 300 mg once or twice per day for 3-6 months.

[0038] As shown below, the pharmaceutical composition of the invention has proven to be surprisingly effective in the treatment of solid cancers. In particular, without being bound to specific theories, the active ingredients of the extract of Sambucus nigra L. petioles and bark, besides having a potent anti-cancer effect, would act in synergy with anti-cancer chemotherapeutics by reducing cell proliferation and inducing apoptosis and cell cycle arrest.

[0039] The data reported herein show that the direct effects of herbal medicine on both cancer development and the development of side effects related to standard chemotherapy in two patients with solid cancers are truly effective.CLINICAL CASE 1

[0040] A 62-year-old woman with no previous health problems has a mass in the retroareolar region of the left breast. The patient had an ECOG (Eastern Cooperative Oncology Group) performance status with no prior oncology history and no previous breast or lung disease. Physical examination revealed a small, soft, moving nodule with no associated skin changes in the retroareolar left breast mass, but there was minimal nipple narrowing. The nodule had appeared 3 years earlier during breast screening and had been misinterpreted as a benign finding. As a matter of fact, in this region of the left breast, the ultrasound performed three years earlier had shown a 5 mm lesion evaluated as category 2 of the Breast Imaging-Reporting and Data System (BI-RADS). Mammography was also insignificant due to its superficial position, under the retroareolar skin and due to its small size. Regular radiological follow-up and clinical observation were recommended. However, after this period, the lesion increased in size and became palpable, and the patient was first imaged by conventional means. Digital mammography showed a single, oval, high density lesion with well-defined margins without microcalcification that matched the palpable finding; it was also associated with nipple narrowing. The ultrasound examination confirmed a solid nodule (measuring 27 mm x 10 mm), highly vascularized to color Doppler, oval in shape, well-defined margins, and a slightly heterogeneous echostructure. The lesion extended to the overlying subcutaneous adipose tissue without any acoustic shadow. The results were suggestive of a neoplasm without clear evidence of a benign or malignant nature. No suspicious axillary lymph nodes were found during ultrasound examination. Fine needle guided aspiration showed no malignant cytological characteristics. The fine needle aspiration biopsy was also unsatisfactory for diagnosis. Therefore, and due to the highly suspected clinical characteristics of the breast lesion (nipple alteration and increased size), the patient underwent a partial diagnostic breast resection which demonstrated a malignant lesion compatible with a neoplasm originating from smooth muscle tissue compatible with leiomyosarcoma. Pathological evaluation revealed malignant mesenchymal neoplasm consisting of pleomorphic fused cells with high mitotic rate [up to 5 mitoses / 10 high potency fields (HPF)] and proliferation index (Ki67: 50%), which expresses smooth muscle markers consistent with leiomyosarcoma.

[0041] Based on histological diagnosis, a staging CT scan was performed which confirmed the presence of a mass of soft tissue in the left breast (approximately 3 cm in the larger diameter) without invasion of the chest wall and a suspicious pulmonary nodule in the left lower lobe (approximately 2 cm). CT scan images showed uneven-edged mass with uneven contrast; there were no lymph nodes with radiological malignancy characteristics. No abdominal lymphadenopathies, peritoneal or hepatic metastases were identified.

[0042] In order to discriminate between a primary lung lesion and a single lung metastasis from breast leiomyosarcoma, a preoperative Positron Emission Tomography (PET / CT) was performed which showed a large abnormal fludeoxyglucose 18F (FDG) uptake in the lower left lung lobe corresponding to CT scan findings and a lesion in the left breast.

[0043] Both PET / CT and chest-abdominal CT scan did not reveal any further pathological findings. Therefore, the patient underwent bronchial fibroscopy with aspirate and bronchial lavage, both negative for neoplasm.

[0044] A CT-guided fine needle biopsy of the pulmonary nodule was performed which revealed a primary pulmonary adenocarcinoma on microscopic examination. The patient was in good general health, laboratory tests were normal and the pre-surgical work-out showed no contraindications to major thoracic surgery associated with homolateral breast conservative surgery.

[0045] The patient underwent a curative lower left lobectomy and lymphadenectomy by an anterolateral thoracotomy and at the same time as a left central quadrantectomy by removing the previous incision and the nipple / areola complex (NAC). Surgical margins were determined by macroscopic and histological examination of frozen sections of breast specimens in the operating room. An adequate safety margin of 1 cm was obtained. Since lymphatic diffusion and lymph node metastases are not typical characteristics of leiomyosarcomas, no sentinel lymph node biopsy or axillary lymph node dissection were performed.

[0046] The postoperative course was smooth and the patient was discharged on the fourth day after surgery. A 2-centimetre subpleural nodule was described on macroscopic examination of the pulmonary specimen. No necrosis or vascular invasion was observed under the microscope. All lymph nodes were negative.

[0047] The histological diagnosis was moderately differentiated lepidic adenocarcinoma (G2) (pT2aN0Mx, according to the seventh edition of the TNM) of the lung, with focal invasion of the visceral pleura. The histological diagnosis of the breast lesion was high grade leiomyosarcoma.

[0048] Treatment: Radical surgical therapy with adjuvant radiotherapy to prevent local recurrence of the breast cancer and taking Sambucus nigra L. one 670 mg tablet (phytocomplex total dry extract) twice a day for six months.

[0049] Follow-up through contrast-enhanced CT scan, complete abdomen ultrasound and mammography after 5 years shows that the patient is healthy and disease-free.CLINICAL CASE 2

[0050] 70-year-old male, free from any other disease, with T3B N1 MO prostate cancer diagnosed by means of full abdominal ultrasound + contrast enhanced total body CT scan, bone scan, biopsies by means of transrectal ultrasonography (Adenocarcinoma Gleason score 4+3). PSA 27.

[0051] Treatment: ENANTHONE 11.25 mg / ml monthly for 24 months and taking Sambucus nigra L. 1 , 670 mg tablet (phytocomplex total dry extract) twice a day for six months.

[0052] Follow-up by means of contrast-enhanced CT scan, upper and lower abdomen ultrasound and transrectal ultrasonography at 5 and 10 years has shown complete remission of the disease (TO NO), PSA 2, good general conditions.

[0053] The synergy between black elderberry and enanthone resulted in the patient with prostate adenocarcinoma to be disease-free at 10 years and in the patient with breast leiomyosarcoma plus synchronous homolateral pulmonary adenocarcinoma at 5 years of follow-up to be free from recurrence and / or lymph node metastases and in the long term, even with pharmacological treatment solely based on total dry extract of elderberry.

Claims

CLAIMS1. Pharmaceutical composition comprising total dry extract of elderberry petioles and bark for use in the prevention and treatment of tumours.

2. Pharmaceutical composition according to claim 1 , comprising said extract combined with an extract of elder flowers and / or berries.

3. Pharmaceutical composition according to claim 1 or 2, further comprising total dry extract of pomegranate.

4. Pharmaceutical composition according to any one of claims 1 to 3, further comprising an anticancer drug selected from enanthone, Docetaxel, Folfiri, Oxaliplatin, Capecitabine, Gemcitabine, monoclonal antibodies.

5. Pharmaceutical composition according to any one of claims 1 to 4, wherein the total dry extract of petioles and bark comprises polyphenols, glycoside phenols, anthocyanins and [3-carotene.

6. Pharmaceutical composition according to claim 5, wherein said polyphenols comprise 4-caffeoyl quinic acid, 3-caffeoyl quinic acid, caffeic acid, catechin, epicatechin, rutin, 3-coumaroyl quinic acid, isorhamnetin-3-rutinoside, ferulic acid, kaempferol-3 rutinoside, 1 ,5-dicaffeoylquinic acid, said phenols comprise sambunigrin, prunasin, zierin and olocalin, said anthocyanins comprise cyanidin-3- sambubioside, cyanidin-3-glucoside, cyanidin-3-monoglicoside an cyanidin-3,5- diglucoside.

7. Pharmaceutical composition according to claim 5 or 6, wherein said polyphenols are present in amounts comprised between 4.800 ppm and 10.900 ppm on the dry weight of the total extract of petioles and bark, said phenols are present in amounts comprised between 5.000-20.000 ppm, said anthocyanins are present in amounts of about 300.000 ppm, said [3-carotene is present in an amount of comprised between 5 ppm and 12 ppm.

8. Pharmaceutical composition according to any one of claims 1 to 7, for use in the treatment of cancer preferably solid cancers, comprising the administration of the total dry extract of elderberry petioles and bark in an amount comprised between 300 mg and 1500 mg once or twice per day for 1 -10 months.

9. Pharmaceutical composition according to claim 8, further comprising the administration of a dry extract of elder berries in an amount comprises between 100 mg and 600 mg once or twice per day for 1 -10 months.

0. Pharmaceutical composition according to claim 8 or 9, further comprising the administration of the dry extract of pomegranate in an amount comprised between 100 mg and 600 mg once or twice per day for 1 -10 months.

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