病情分析：你好，看你说的情况，那看你这时有过高危的性行为的，那看你在四周时检测结果为阴性，那说明是没有感染的意见建议：而现在看艾滋病的窗口期是一个月的时间，那你要是不放心，那在六周后在复查一次也行，如果说这次检测结果还是阴性，那就可以说是排除有感染了anti-hiv是什么意思
厚渺酥4044
Anti-HIV是指人类免疫缺陷病毒抗体筛查
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anti-hiv参考值阴性，结果可疑待检查什么意思
anti-hiv参考值阴性，结果可疑待检...
病情描述（发病时间、主要症状、症状变化等）：anti-hiv参考值阴性，结果可疑待检查什么意思曾经治疗情况和效果：是，第一次在医院检查，出现这样的症状想得到怎样的帮助：这样的情况感染的几率大吗
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因不能面诊，医生的建议仅供参考
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问题分析：这样的情况感染的几率大不大不好说也不能说有感染。意见建议：一般的情况下会让你重新再次验血之后，看这一次的结果。
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专长：外科
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问题分析：根据你现在的这个检查结果来看，通常来说，你现在的这个情况基本上来说已经可以排除艾滋病等情况了意见建议：你现在这个情况通常来说感染的可能性不会很大的啦，血液的检查指标通常来说还是比较可靠的，那如果现在这几天出现了全身淋巴结肿大等情况最好再考虑复查
问ALT是什么意思
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指导意见：你，可能肝功能指标，这种情况一般及时休息，继续的保养，你的指标是正常的，就不要担心，问题不大的啊。
问科检查的报告单显示排卵期可疑5.0-25.0黄体期妊辰大于...
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病情分析： 　　　女性的排卵日期一般在下次月经来潮前的14天左右。卵子自卵巢排出后在输卵管内能生存1～2天，以等待受精；男子的精子在女子的生殖道内可维持2～3天的生命力，故在卵子排出的前后几天里性交容易受孕。意见建议：为了保险起见，我们将排卵日的前5天和后4天，连同排卵日在内共10天称为排卵期。因为在排卵期内性交容易受孕，所以排卵期又称为易受孕期或危险期。
问检验报告弓型虫lgM抗体定量7.80（可疑）参考值&6.。...
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病情分析： 治疗上主要为抗炎，首先得弄清病因，其次才谈得上治疗。希望你去正规的医院做相关的检查，弄清楚后在进行系统正规的治疗。
问甲状腺峡部低回声结节————淋巴结可疑什么意思啊
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问题分析：你好，据你的情况描述来看和颈部甲状腺b超来看，你属于甲状腺结节，可以通过手术治疗来切除结节，做病理，之后来看是哪种定性物质。意见建议：建议，如果有什么不适建议到正规医院耳鼻喉科进行系统专科检查和治疗，据你的具体情况制定个体化治疗方案，可以通过手术来切除甲状腺结节，淋巴结可疑是指，可能淋巴结处有转移灶或者是淋巴结肿大，需要病检来，确诊。祝早日康复。
问乳酸血症可疑，半乳糖略高什么意思啊
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半乳糖略高,未发现其他代谢异常产物.半乳糖略高可以是食用乳类制品引起的生理性改变,请结合临床上有无消化系统症状综合考虑.
问HCG，尿绒毛膜促性腺激素，可疑，阴性。是什么意思
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你好！需要再延迟一周后做血HCG检查确定是否怀孕，孕6-8周后做子宫附件B超检查排除宫外孕
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评价成功！Anti-HIV agents
United States Patent 4985249
Anti-HIV agent containing as active components alkaline water extracts of pine cone and method for producing high molecular weight substance having anti-HIV activity.
Inventors:
Sakagami, Hiroshi (c/o Medical Department, Showa University of 1-5-8, Hatanodai, Shinagawa-ku, Tokyo, JP)
Konno, Kunio (c/o Medical Department, Showa University of 1-5-8, Hatanodai, Shinagawa-ku, Tokyo, JP)
Nonoyama, Meihan (c/o Medical Department, Showa University of 1-5-8, Hatanodai, Shinagawa-ku, Tokyo, JP)
Application Number:
Publication Date:
01/15/1991
Filing Date:
06/22/1988
Export Citation:
SAKAGAMI; HIROSHI
KONNO; KUNIO
NONOYAMA; MEIHAN
Primary Class:
Other Classes:
International Classes:
A61K36/15; (IPC1-7): A61K35/78
Field of Search:
424/195.1, 514/54, 514/934
View Patent Images:
&&&&&&PDF help
Other References:
Chem. Abst. 110(9): 69032u, 1989.
Chem. Abst. 109(15): 1.
(1) "Partial Purification of Various Active Substances from Pine Cone Extract of Pinus Parviflora Seib. et Zucc" (abst. 109: 125935j) (published on Dec. 28, 1987).
(2) "Spontaeous Production of Differentiation-Inducing Factor(s) by Mouse Macrophage-Like Cell Line" (abst. 110; 69032u) (published Aug. 28, 1988).
Biological Abstracts, vol. 81, No. 12, 1986, No. 113512, Sakagami et al.
Chemical Abstracts, vol. 78, No. 19, 14th May 1973, p. 70, No. 119653r, Joubert et al.
Primary Examiner:
Rollins, John W.
Attorney, Agent or Firm:
Oliff & Berridge
What is claimed is:
1. Anti-HIV agents containing as active components extract which has been extracted with alkaline water from pine cone.
2. Anti-HIV agents as claimed in claim 1, wherein said extract is extracted from Pinus parviflora Sieb. et Zucc pine cone.
3. Anti-HIV agents as claimed in claim 1, wherein the active components are at least one member selected from the group consisting of KS-6 and KS-7 which are contained in the extract which has been extracted with alkaline water from pine cone.
4. Anti-HIV agent as claimed in claim 3, wherein said member is KS-6, which has a brownish color with an absorption soulder at 260-280 nm, which dissolves in water, and mixtures of water and alcohol or acetone, and is composed of 43.2% wt% arbon, 4.0 wt % hydrogen, 2.6 wt% nitrogen, and has a molecular weight of about 10 kD by gel filtration under alkaline condition, said KS-6 being obtained from the precipitate from alikaline-water extract of pine cone at pH 5.
5. Anti-HIV agent as claimed in claim 3, wherein said member is KS-7, having a brownish color with an absorption shoulder at 260-280 nm, capable of being dissolved well in water, but tending to precipitate in physiological saline and mixtures of water and alcohol or acetone, and is composed of 33.5 wt% carbon, 4.23 wt 5% hydrogen and undetectable amount of nitrogen and sulfate, and having a molecular weight of 10-200 kD under alkaline condition by gel filtration, said KS-7 being from recovered from supernatant fraction of alkaline water extracts by ethanol precipitation after adjustment to pH 5.
6. The anti-HIV agent of claim 1 wherein said extract has been extracted with alkaline water having a pH of at least 8.
7. The anti-HIV agent of claim 6 wherein said alkaline water has a pH of from 8 to 13.
8. A method for producing KS-6 comprising the steps:
extracting a pine cone with 1% NaOH after removal of alcohol and hot water extractable materials, precipitating the NaOH extract at pH 5, predissolving the precipitate by 1% NaOH to remove impurities and extensively dialyzing against distilled water.
9. An anti-HIV composition containing an effective amount of KS-6 as obtained according to claim 8, and a suitable carrier or diluent.
10. A method for producing KS-7 comprising the steps:
extracting a pine cone with 1% NaOH after removal of alcohol and hot water extractable materials,
precipitating the NaOH extract with an equal volume of ethanol, and
extensively dialyzing the precipitate against water.
11. An anti-HIV composition containing an effective amount of KS-7 obtained according to claim 10, and a suitable carrier or diluent.
Description:
BACKGROUND OF THE INVENTIONThe present invention relates to plant-derived high molecular weight substances which have potent anti-HIV activity. The human immunodeficiency virus (HIV) is the causative agent associated with AIDS (acquired immune deficiency syndrome), ARC (AIDS-related complex) and related diseases. A distinguishing feature of HIV is its selective cytotoxicity for helper T lymphocytes. Severe and diverse aberrations of the immune system significantly reduce the host defense against various opportunistic infections, resulting finally in a host death. Nowadays, many anti-HIV agents including Krestin and Lentinan have been isolated from plants and chemically synthesized. However, the effect of most of these agents are generally weak and they have severe side effects. In order to explore new type of anti-HIV substance, the present inventors have compared the anti-HIV activity of various plant components. The present inventors have found potent anti-HIV activity in the antitumor substances obtained from aqueous alkaline extract of pine cone. The substances with the most potent anti-HIV activity was named as KS-6 and KS-7. SUMMARY OF THE INVENTION An object cf the present invention is to provide two plant-derived high molecular weight substances which have potent anti-HIV activity. Another object of the present invention is to provide anti-HIV agents having as active components the above-mentioned high molecular weight substances. Still another object of the present invention is to provide a method for obtaining the high molecular weight anti-HIV substances from pine cones. The anti-HIV agents in the present invention are characterized in that said agents which contain alkaline-water extracts of various pine cones such as Pinus parviflora Sieb. et Zucc., especially high molecular weight anti-HIV substances of this extract. The high molecular weight anti-HIV substances described above can be obtained by extraction of the pine cone with alkaline water. Other objects and characteristics of the invention will become apparent from further disclosure of the invention to be made in the following detailed description of preferred embodiment, with reference to the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows dose-dependent inhibition by KS-7 of viral production in CEM cells chronically infected with HIV. FIG. 2 shows time course of inhibition by KS-7 of viral replication in CEM cells chronically infected with HIV. FIG. 3 shows effect of KS-6 added just after the HIV infection. FIG. 4 shows effect of pretreatment wIth KS-6 or KS-7 on HIV replication.
DETAILED DESCRIPTION OF THE INVENTION Pine cones of various Pine trees such as Pinus parviflora Sieb. et Zucc., Pinus densiflora Sieb. et Zucc., and Pinus thunbergii Parl., can be used for the preparation of anti-HIV substances. It is preferable to use Pinus parviflora Sieb. et Zucc. due to an of active substances. Pine cones collected at summer season are enriched in floating fatty materials, which might disturb further purification. Therefore the pine cones should be collected in October when active substances accumulate in abundance. The substances with a certain extent of activity can be extracted from pine cone by hot water extraction. However alkaline water extraction is more effective than hot water extraction to obtain larger amounts of the active substances the pine cone. Either alkaline extracts or further fractionated materials can be used as effective anti-HIV substances. The present inventors presumed that substances which are contained in this extract and used as active ingredients are high molecular weight substances. The most potent substances were named as KS-6 and KS-7. Alkaline water extracts used in this invention can be prepared by directly extracting a pine cone with aqueous alkaline solution such as aqueous NaOH. It is preferable, however, to obtain alkaline water extracts from a pine cone in which oils and hot-water extractable materials have been removed by extensive alcohol and boiling water washing before alkaline water extraction. Either organic bases or inorganic bases can be used as alkaline substances for extraction media. The pH in aqueous alkaline solution is preferable to be more than 8.0; most preferably pH being 9.0 to 13.0. The extracted solution should be concentrated to desired concentration, if needed, neutralized with appropriate acid and then lyophilized to give appropriate concentrated solution or solid. These can be used as active components without further fractionation. Alternatively, the concentrate can be separated into precipitate and supernatant by some appropriate methods such as centrifugation. KS-6 can be obtained from precipitate by reextraction with alkaline water, and KS-7 from supernatant by ethanol precipitation. The present invention will be explained by the following one example, however, the invention will not be restricted to this example. PURIFICATION PROCEDURE OF KS-6 AND KS-7 500g of pine cones was washed succesively, twice with about 5 liters of methanol, and twice with 85% ethanol for 4 hours with reflux, and then extracted for 6 hours, 3 times with boiling water. The residue was extracted twice with 5 liters of 1% sodium hydroxide aqueous solution for 6 hours at room temperature. After removal of insoluble materials by filtration through gauze, the pH of the filtrate was adjusted to 5.0 with acetic acid. The precipitate was collected by centrifugation for 20 minutes at 10000 xg at 4° C., dissolved in a small volume of 1% sodium hydroxide aqueous solution, centrifuged to remove insoluble materials and the supernatant was neutralized with acetic acid. The obtained substance is KS-6. The remaining supernatant was precipitated with one volume of ethanol to obtain KS-7. both KS-6 and KS-7 were dialyzed against distilled water and lyophilized. Yield of these substances KS-6 and KS-7 was about 0.5% and 1%, respectively. ANALYTICAL PROCEDURES Neutral sugar composition was analyzed by gas-liquid chromatography of trimethylsilyl derivatives of methyl glucosides obtained by methanalysis in methanolic HC1 for 16 hours at 65° C. Neutral sugar content was also determined by the phenol-sulfuric acid method. Uronic acids were analyzed by gas-liquid chromatography of trimethylsilyl derivatives after their conversion to aldonolactone. Uronic acids content was determined by the carbazoIe method. Carbon, hydrogen, nitrogen and sulfur content was determined by element analyzer. ENDOTOXIN ASSAY The presence of endotoxin was determined by the Endospecy (Endotoxin Specific Test, Seikagaku Kogyo, Ltd., Japan) with Escherichia coli 0111:B4 endotoxin as a standard. HIV INFECTION TO T CELL The CEM cell used was a T cell line derived from human leukemia patient. The virus used was HIV N.Y. strain. CEM cells were infected by addition with HIV-carrying culture medium. Infectivity of the virus released into the culture medium was assayed with reverse transcriptase (RT) activity and p24 antigen capture method. KS-6 has a dark brownIsh color, with an absorption shoulder at 260-280 nm. It easily dissolves in water, and mixtures of water and alcohol, or acetone. It contains carbon (43.2wt %), hydrogen (4.0wt %), nitrogen (2.6wt %) and no sulfur. KS-6 contains also neutral sugars (11.0wt %) consisting of fucose (9.4mol %), mannnose (19.0mol %), galactose (44.7mol %) and glucose (26.9mol %), and a negligible amount of uronic acid (1.7wt %). The molecular weight determined by gel filtration under alkaline condition was 10 kD. KS-7 also has brownish color, with an absorption shoulder at 260-280 nm. It easily dissolves in water, but tends to precipitate in physiological saline and the mixtures of water and alcohol, or acetone. It has carbon (33.55wt %), hydrogen (4.23wt %), and undetectable amount of nitrogen and sulfur. KS-7 contains also neutral sugars (39.5wt %) consisting of arabinose (16.5mol %), mannose (16.2mol %), galactose (39.3mol %) and glucose (26.0 mol %), and abundant uronic acid (58.2wt %). The contamination of endotoxin was around 0.0025%. The molecular weight determined by gel filtration under alkaline condition was 10-200 kD. ANTI-HIV ACTIVITY OF KS-6AND KS-71. Effect of KS-7 on chronic HIV infection A clone of the CEM T cell line was cultured for long period with HIV at low virus infection ratio. The chronically infected cells were incubated for 1 week with various concentration of KS-7 (0-350 μg/ml). At various time points thereafter HIV production in culture supernatants was measured by RT method. As shown in FIG. 1, HIV replication was reduced by 66% by addition of KS-7 at concentration of as low as 7 μg/ml. KS-7, at concentration of more than 70 μg/ml, inhihIt HIV replication by more than 85%. Viability of CEM cells was not affected by treatment with 70 μg/ml of KS-7. A similar degree of inhibition of HIV replication was observed in cells treated with 200 μg/ml suramine, a potent inhibitor of retrovial RT. However, this concentration of suramine reduced the viability of CEM cells to 78%. The time course of the inhibition of HIV replication by KS-7 was next investigated. The chronically infected CEM cells were treated with 50 pg/ml or 100 μg/ml KS-7 for 5, 7, 10, or 17 days and the viral RT activity recovered from the culture supernatant was determined as shown in FIG. 2. RT activity found in supernatants of the treated cells was at any time points inhibited by about 90%. This indicates that KS-7 inhibit viral production. It should be noted that the cells were diluted 1:5 and supplemented with fresh medium containing KS-7 at the time indicated by arrows, and the RT activity (ordinate) was plotted in log scale in FIG. 2.2. Effect of KS-6 on acute HIV infection Next, the ability of KS-6 to inhibit virus replication in acute infection with HIV was investigated. The CEM cells were infected with HIV at high virus concentration. After viral adsorption, the cells were washed 3 times with medium and maintained at 5×105 cells/ml in medium supplemented with various concentrations of KS-6 (ranged from 0-30μg/ml). After incubation for 3 to 7 days, concentration of HIV p24 antigen in the culture supernatants was determined by a commercially available p24 antigen capture assay (Abbot's Lab.). As shown in FIG. 3, the antiviral activity was induced by KS-6 at more than 0.3 μg/ml. Higher concentration of KS-6 ("3 pg/ml" or "30 μg/ml") inhibited HIV replication by 50% and 88%, respectively. The viability of CEM cells was not significantly affected at any of these concentrations.3. Effect of CEM cell pretreatment by KS- 6 or KS-7 It was Investigated whether pretreatment of CEM cells with KS-6 or KS-7 could enhance the inhibition of HIV replication. CEM cells were Pretreated for 1 or 7 days with various concentrations of KS-6 (0.3-30 μg/ml) or KS-7 (3-100 μg/ml). After washing, the cells were infected with HIV virus at high virus concentration, and maintained in medium containing the same concentrations of KS-6 or KS-7 used in pretreatment. After 5 days incubation, the concentratIon of p24 in culture supernatant was determined by p24 antigen capture method. As seen in FIG. 4, a reduction of viral titer was observed in CEM cell cultures pretreated with 0.3 μg/ml of KS-6. An 80-95% reduction of HIV replication was attained by KS-6 at the concentrations of 3 and 30 μg/ml, respectively. 1n contrast, antiviral activity of KS-7 was slightly weaker than that of KS-6. The anti-HIV activity of KS-7 was detected at more than 10 μg/ml, and 95% inhibition of HIV replication was achieved by KS-7 at 30-100 μg/ml. The present data demonstrate that antitumor substances KS-6 and KS-7, prepared from alkaline water extracts of pine cone effectively inhibit HIV replication, and the antivial activity of these agents is augumented by pretreatment of the target cells with these agents before HIV infection. A distinguishing feature of human immunodeficiency virus (HIV) is its selective cytotoxicity for helper T lymphocytes. Infection in vivo is manifested by severe and diverse abberations of immune system and causes death. As descrived above, KS-6 and KS-7 of the present invention have potent anti-HIV activity, as well as their immunopotentiating activity manifested by activation of human peripheral blood monocytes and polymorphonuclear cells. Therefore, it is highly probable that these agents might improve the condition of AIDS patients and its effect might be augumented by combinational treatment with other chemotherapeutic agents (e.g. azido-thymidine, dideoxycytidine, suramine).
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