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Sambucus nigra L. Scientific Name: Sambucus nigra L. Synonyms: Sambucus nigra var. canadensis (L.) B. L. Turner; Sambucus nigra subsp. canadensis (L.) Bolli Family: Adoxaceae Genus: Sambucus Species: nigra Common name: Black Elder, Elder tree, Black-berried European Elder, Boor Tree, Bore Tree, Bountry, Common Elder, Ellanwood, Ellhorn, European Elder, German Elder, Holunder, Pipe Tree Parts used: Bark, leaves, flowers, berries Plant description: Sambucus nigra L. is a deciduous shrub or small tree growing to 6 m (20 ft) tall and wide (rarely 10m tall). The bark, light grey when young, changes to a coarse grey outer bark with lengthwise furrowing. The leaves are arranged in opposite pairs, 10–30 cm long, pinnate with five to seven (rarely nine) leaflets, the leaflets 5–12 cm long and 3–5 cm broad, with a serrated margin. The hermaphrodite flowers are borne in large, flat corymbs 10–25 cm diameter. The individual flowers are ivory white, 5–6 mm diameter, with five petals. The fruit is a glossy dark purple to black berry 3–5 mm diameter. Constituents: Hyperoside, quercetin, iso-quercetin, rutin, cyanidin-3-glucoside, cyanidin-3-sambubioside, sambu-nigrin, vitamins A, B1, B2, B6, C, and P. minerals, lectin, choline, tannin, lupeol, β-sitosterol, beltulin, potassium nitrate, holocalin, prunasin, zierin.

Reported chemical constituents present in Sambucus nigra L.

Hyperoside Quercetin Iso-quercetin

Rutin Cyanidin-3-glucoside Cyanidin-3-sambubioside

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Sambunigrin Vitamin A Vitamin B2

Vitamin B1 Vitamin B6

Vitamin C Vitamin P Choline

Lupeol β-sitosterol Beltulin

Potassium nitrate Prunasin Holocalin Zierin

Medicinal uses: diuretic, cathartic, emetic, aperient, purgative, alternative, expectorant, diaphoretic, laxative

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Actions of the herb: Builds and strengthens the damaged blood vessels, hemostatic, nerve disorders, neuralgia, epilepsy, laxative (for small children irritated and inflamed intestines), constipation, reduces inflammation of the urinary tract and bladder, kidney problems, urinary problems, increasing the production of urine, eliminating excess water from the body, arthritic and rheumatic complaints, nasal or bronchial catarrh, relieves asthma, bronchitis, sore throat, fighting influenza and colds as an excellent internal cleanser, tonsillitis, back pain, induces perspiration to reverse the effects of a chill lumbago, sciatica, fevers inducing sweat, obesity, cleansing the body. Externally the flowers have been used: as an excellent eye wash when strained, especially for conjunctivitis, for freckles, for rashes, for skin ailments, especially dry skin for sunburn, in skin washes to refine the complexion, to heal burns, to help relieve acne, eczema, psoriasis to relieve headache (compress) used externally for burns chapped skin (the oil) cuts, scalds, scrapes, swelling, diaphoretic, diuretic, astringent, emetic, anti-viral agent, Herpes virus infection. It has immune-modulating, anti-oxidant and insulin-stimulating properties (Diabetes), stress, Herpes simplex and HIV treatment. Side effects and toxicity: The consumption of uncooked berries or juice can result in vomiting and diarrhea. Certain constituents of the leaves, stems, flowers, and roots contain poisonous alkaloids. It has also been reported that small percentages of the general population have a type-1 allergy to S. nigra as evidenced by positive-skin prick or RAST test. Incompatibilities with other medications Medications that decrease the immune system (Immuno-suppressants) interact with elderberry. Elderberry can increase the immune system. Taking elderberry along with some medications that decrease the immune system might decrease the effectiveness of medications that decrease the immune system. Some medications that decrease the immune system include azathioprine (Imuran), basiliximab (Simulect), cyclosporine (Neoral, Sandimmune), daclizumab (Zenapax), muromonab-CD3 (OKT3, Orthoclone OKT3), mycophenolate (CellCept), tacrolimus (FK506, Prograf), sirolimus (Rapamune), prednisone (Deltasone, Orasone), corticosteroids (glucocorticoids), and others. Dosage Adolescents over 12 years of age, adults, elderly; Herbal substance for tea preparation: 2-5 g 3 times daily. A) Liquid extract: 3-5 ml 3 times daily. B) Tincture: 10-25 ml 3 times daily. The use is not recommended in children under 12 years of age. Range of Application Not to be taken for more than 1 week. If the symptoms persist during the use of the medicinal product, a doctor or a qualified health care practitioner should be consulted. Contraindications Pregnancy, lactation and hypokalaemia. Chemical Identification Tests of S. nigra The extract of S. nigra in chemical colour reaction exhibited the presence of tannins, saponins, carbohydrates, sterols and steroids. Microscopic examination of S. nigra flower The microscopic diagnostic features include small oil globules. An upper epidermis is lined with cells that have slightly thickened and beaded walls along with striated cuticle. Calyx has epidermal cells with striated walls and cells at the basal surface revealed unicellular, marginal teeth. Calcium oxalate crystals and numerous pollen grains were also found. Pollen grains are sub-spherical and smooth with furrows and pores (Wallis 2005; Evans 2009) see figure below.

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Figure: Sambucus nigra and its diagnostic features

Thin-layer Chromatography of S. nigra S. nigra extract in chloroform – methanol – water (80:20:2) solvent system the Rf values of the spots at 254nm were 0.04, 0.08, 0.12, 0.16, 0.25, 0.35, 0.42, 0.49, 0.71 and at 366nm were 0.01, 0.05, 0.09, 0.14, 0.23, 0.52, 0.87. In ethyl acetate – methanol – water (100:16.5:13.5), the Rf values at 254nm were 0.01, 0.08, 0.15, 0.22, 0.28, 0.33, 0.43, 0.52, 0.59, 0.69, 0.76, 0.81 and at 366nm was 0.09, 0.16, 0.38, 0.45, 0.61. Fourier Transform Infra-red Spectroscopy of S. nigra Extract of S. nigra in FT-IR spectrum displayed peaks at: 3300 (OH), 2908.97 (C-H), 1601.36 and 1519.64 (aromatic ring), 1045.63 (C-O-C) cm-1.

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Insecticidal activity of S. nigra No mortality was seen in doses of 1-50 mg; while at the dose of 100mg the mean mortality time was found 10.4±2.63. Anthelmintic activity of S. nigra Earth worms responded stimulation at all doses of S. nigra from 5 to 25mg in 3-5 hours of drug exposure. The mortality time in helminthes were found 24±0 on exposure to 10 mg and 25 mg extract of S. nigra, respectively. In 25mg of dose death occur with hemorrhage. Molluscicidal activity of S. nigra No reduced activity was noted at the end of 72 hours in the snails exposed to 1 and 25mg of S. nigra extract. Dose and time dependent decrease of activity was observed in 50 and 75mg doses of the drug. 4 out of 6 snails were found dead at the end of 72 hours in snails given 100mg of S. nigra extract. Pronounced paralytic effect was seen at the end of 48 hours in the snails exposed to 500mg of the drug, whereas in those exposed to 1000 mg; paralytic effect was noted at the end of 24 hrs. Fatality was seen at the end of 72 hours in the snails exposed to 500mg of S. nigra extract. On exposing to 1000 mg of the drug, paralytic time was recorded as 24±0, and mortality 48±0. Anti-bacterial activity of S. nigra S. nigra showed 20±2 zone of inhibition against A. hydrophila (gram-negative bacteria) as compared to the control drug, Gentamicin. MIC of S. nigra extract was observed at 12mg/ml and 22mg/ml, against S. epidermidis and S. pyogenes (gram-positive bacteria) respectively. Anti-fungal activity of S. nigra S. nigra extract did not exhibit any zone of inhibition against pathogenic fungi used in experiment. In MIC test of S. nigra extract no inhibitory activity was recorded against all the tested pathogens used in experiment. Anti-oxidant activity of S. nigra S. nigra, (100mg) showed highest DPPH scavenging activity 76.24%, and total antioxidant activity at 100 mg, 68.91%.

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Effects of S. nigra on locomotor and exploratory behavior of mice The anxiolytic effect was assessed by using open field, head dip, light and dark, cage cross and swimming activity. At the dose of 100mg/kg of S. nigra, CNS inhibitory effects were observed in following activities; in open field activity (75.16±3.68) counts in 30 minutes, in light and dark activity 3.83±0.65 times in cage cross test, 8.33±1.01 times. Whereas, in forced swimming test (FST) the mean force mobility time recorded was 0.24±0.03 seconds, at 300mg/kg dose of S. nigra extract. At 500mg/kg of S. nigra, the mice dipped its head 7.16±0.77 times. Locomotor and exploratory activity was observed substantially reduced in comparison to control and standard drug, Diazepam (2 mg/kg-1). Effects of S. nigra on Analgesic activity using Acetic acid Analgesic activity is widely assessed by acetic acid induced method. The abdominal constrictions were observed. S. nigra at the dose of 50 mg/kg exhibited maximum inhibition of writhes induced by acetic acid in first phase (56.76%) second (11.42%) and third phase (16%). Effects of S. nigra on Analgesic activity using Formalin The results exhibited prominent analgesic effect in comparison to aspirin (orally administered, 300 mg/kg). The analgesic effect of aspirin in phase 1, 2 and 3 was 34.48%, 31.01% and 26.4% respectively. S. nigra showed inhibition of the licking and biting response induced by formaldehyde at the dose of 10mg/kg as follows: 25.16% (first phase), 11.11% (second phase) and 34.57% (third phase). While at the dose of 50mg/kg the percentage inhibition in 1st, 2nd and 3rd phase were observed to be 1.17%, 92.59% and 33.2% respectively. Effects of S. nigra on carrageenan induced anti-inflammatory activity S. nigra (300mg/kg) at 0.5 hour revealed 20.68% paw volume inhibition. Whereas, S. nigra (500mg/kg) exhibited maximum paw volume inhibited 27.42% at 4.5 hours. Aspirin had 22.22% paw volume inhibition at 1.5 hours. Effects of S. nigraon hematological parameters of male rabbit’s blood In male test group treated with S. nigra, hemoglobin (10.808±0.063), hematocrit (37.75±0.0836), MCV (70.25±0.0836), MCH (20.25±0.0836), total leucocyte count (6.723±0.02) and platelet count (590.05±0.32) were found rose. RBC (5.35±0.051) and MCHC (28.835±0.0739) counts were found slightly lowered in the test group as compared to the control group. Effects of S. nigraon hematological parameters of female rabbit’s blood Hematocrit (42.808±0.063), MCH (19.85±0.0836) and MCHC (28.008±0.063) levels were observed slightly lowered; while platelet count (322.5±0.836) was moderately lowered. Hemoglobin (12.183±0.1036), RBC count (6.03±0.0073), MCV (70.75±0.0836) and total leucocyte count (6.25±0.0836) were found elevated in female test group treated with S. nigra as compared to female control group. Effects of S. nigraon Kidney Function Test of male rabbit’s blood Urea (28.5±0.836), serum calcium (15.285±0.0083), total proteins (7.6±0.384), albumin (4.983±0.02) and A/G ratio (1.885±0.0083) were elevated; whereas, creatinine (0.571±0.01), phosphorus (4.625±0.0083), uric acid (0.015±0.002) and globulin (2.65±0.0063) levels were reduced in male test group treated with S. nigra as compared to male control group. Effects of S. nigra on Kidney Function Test of female rabbit’s blood There was a slight raise in the levels of phosphorus (5.425±0.0083), uric acid (0.0267±0.0048) and globulin (2.485±0.0083). However, rest of the kidney function parameters; urea (19.5±0.836), creatinine (0.545±0.0083), calcium serum (14.475±0.0083), total proteins

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(7.48±0.0063), albumin (4.985±0.022) and A/G ratio (2.043±0.018) were lowered in test group treated with S. nigra when compared with the respective female control group. Effects of S. nigra on Cardiac Enzymes of male rabbit’s blood LDH (737.5±0.836) was elevated, while CPK (278.67±0.78) and CK-MB (13.5±1.224) enzymes levels were diminished in male treated group with S. nigra in comparison to male control group. Effects of S. nigra on Cardiac Enzymes of female rabbit’s blood LDH (149.33±0.96), CPK (567.5±0.836) and CK-MB (487.33±0.83) enzymes level were reduced in female test group treated with S. nigra as compared to respective female control group. Effects of S. nigra on Lipid Profile of male rabbit’s blood Triglycerides (159.5±0.836), HDL (8.5±0.836) and VLDL (50.5±0.836) levels were raised while cholesterol (27.5±0.836) and LDL (3.75±0.418) levels were diminished in the male test group treated with S. nigra in comparison to the male control group. Effects of S. nigra on Lipid Profile of female rabbit’s blood Cholesterol (38.5±0.836), triglycerides (289.83±1.036) and VLDL (57.5±0.836) levels were found to be elevated; whereas, HDL (3.25±0.418) and LDL (2.25±0.484) levels were lowered in female test group treated with S. nigra as compared to the female control group. Effects of S. nigra on Liver Enzymes of male rabbit’s blood Direct bilirubin (0.118±0.015) and Gamma GT (13.5±0.836) levels were raised. However, the rest of the liver enzymes; SGOT (14±0.632), total bilirubin (0.175±0.0083), SGPT (31.5±0.836) and alkaline phosphatase (52.5±0.836) levels were found to be lessened as compared to their respective male control group. Effects of S. nigra on Liver Enzymes of female rabbit’s blood Total bilirubin (0.24±0.0063) level was slightly reduced. While the levels of the following enzymes, that is, direct bilirubin (0.14±0.019), SGPT (67.5±0.836), alkaline phosphatase (213.83±1.036) and gamma GT (8.5±0.836) were found to be raised in test group treated with S. nigra in comparison with the respective female control group. Effects of S. nigra on Urine analysis of male rabbits The urine parameters of the male test group were similar to that of its respective male control group. Effects of S. nigra on Urine analysis of female rabbits No change in urine was found before and after drug introduction and are similar to respective female control group. Effects of S. nigra on liver enzymes of rabbits’ injected CCl4 prior to dissection The rabbits treated with S. nigra extract revealed following observations after administration of carbon tetrachloride six hours before taking blood for LFT. Total bilirubin 0.08±0.0065 and direct bilirubin 0.006±0.0018 levels were found lowered. Whereas, SGPT 62±0.632, alkaline phosphatase 113±0.632 and gamma GT levels 9.5±0.836 were found elevated as compared to the control group. Autopsy of male rabbits’ treated with S. nigra In heart and stomach tissues of the male group treated with S. nigra, no significant pathology was observed. Sinusoidal dilation and congestion with diffuse lipofuscinosis was seen in liver tissues while acute tubular necrosis was found in kidney tissues.

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Male liver tissue Male kidney tissue Autopsy of female rabbits’ treated with S. nigra No significant pathology was observed heart and stomach tissues of female group treated with S. nigra. Mild portal inflammation and peri-portal fibrosis with foci of macrovesicular steatosis was found in liver tissues. Patchy chronic nonspecific inflammation and tubular injury was found in kidney tissues.

Female liver tissue Female kidney tissue Effects of S. nigra on Diuretic activity of rats The rats given oral dose of 300 mg/kg of S. nigra extract exhibited pronounce diuretic activity 2.11±0.0033 at the end of 4 hours as compared to the control 0.93±0.0036. Furosemide 10 mg/kg showed diuretic activity 2.52±0.0033. Anti-urolithiasis activity of S. nigra S.nigra extract in all the tested concentrations revealed no anti-urolithic activity. References

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